TOPO XL PCR Cloning Kit - Thermo Fisher...

user guide

For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.

TOPO® XL PCR Cloning Kit Five-minute cloning of long (3–10 kb) PCR products Catalog numbers K4700-10, K4700-20, K4750-10, K4750-20, K7030-20

Publication part number 25-0199

Revision date 9 March 2012

MAN0001691

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Contents

Kit Contents and Storage ................................................................................................................................ 5

Introduction ................................................................................................................. 8 Product Overview ........................................................................................................................................... 8

Methods ..................................................................................................................... 10 Produce PCR Products .................................................................................................................................. 10

Gel Purify PCR Products .............................................................................................................................. 12

TOPO® Cloning .............................................................................................................................................. 16

Transform Competent Cells ......................................................................................................................... 17

Analyze Transformants ................................................................................................................................ 20

Appendix ................................................................................................................... 22 Map of pCR-XL-TOPO® ................................................................................................................................ 22

Troubleshoot .................................................................................................................................................. 23

Recipes ............................................................................................................................................................ 24

Accessory Products ....................................................................................................................................... 25

Technical Support .......................................................................................................................................... 26

Purchaser Notification .................................................................................................................................. 27

References ....................................................................................................................................................... 28

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Kit Contents and Storage

Shipping and storage

The TOPO® XL PCR Cloning Kit is shipped on dry ice. Each kit contains two boxes and a plastic bag. Store items as listed in the following table:

Box/Bag Reagents Storage

Box 1 TOPO® XL PCR Cloning reagents Gel Purification reagents

−30°C to −10°C

Box 2 One Shot® Kit −85°C to −68°C

Bag S.N.A.P.™ purification columns and collection vials Gel Purification reagents

Room temperature

(15°C to 30°C)

TOPO® XL PCR Cloning Kits

TOPO® XL PCR Cloning Kits are available with the following One Shot®

Chemically Competent and Electrocompetent Cells (see page 5 for the genotypes of the strains).

Catalog no. Reactions One Shot® Cells Type of Cells

K4700-10 10 TOP10 Electrocompetent

K4700-20 20 TOP10 Electrocompetent

K4750-10 10 TOP10 Chemically competent

K4750-20 20 TOP10 Chemically competent

K7030-20 20 Mach1™-T1R Chemically competent

Genotypes of E. coli strains

TOP10: Use this strain for general cloning and blue/white screening without IPTG.

F- mcrA Δ(mrr-hsdRMS-mcrBC) Φ80lacZΔM15 ΔlacX74 recA1 araD139 Δ(ara-leu)7697 galU galK rpsL (StrR) endA1 nupG

Mach1™-T1R: Use this strain for general cloning and blue/white screening without IPTG. Strain is resistant to T1 bacteriophage.

F- φ80(lacZ)ΔM15 ΔlacX74 hsdR(rk-, mk

+) ΔrecA1398 endA1 tonA (confers resistance to phage T1)

Product use For research use only. Not intended for any animal or human therapeutic or diagnostic use.

Continued on next page

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Kit Contents and Storage, Continued

TOPO® XL PCR Cloning reagents

TOPO® XL PCR Cloning reagents (Box 1) are listed in the following table. Note that the user must supply the thermostable polymerase. Store Box 1 at −30°C to −10°C.

Item Composition Amount for

10 Reaction Kit Amount for

20 Reaction Kit

pCR-XL-TOPO® 10 ng/μL plasmid DNA in: 50% glycerol 50 mM Tris-HCl, pH 7.4 (at 25°C) 1 mM EDTA 2 mM DTT 0.1% Triton® X-100 100 μg/mL BSA phenol red

11 μL 2 × 11 μL

50 mM dNTPs 12.5 mM dATP 12.5 mM dCTP 12.5 mM dGTP 12.5 mM dTTP neutralized at pH 8.0 in water

10 μL 2 × 10 μL

6X TOPO® Cloning Stop Solution

0.3 M NaCl 0.06 M MgCl2

25 μL 2 × 25 μL

XL Control PCR Template 25 ng/μL in TE Buffer 10 μL 2 × 10 μL

XL Control PCR Primers 0.2 μg/μL in TE Buffer (0.1 μg/mL each)

10 μL 2 × 10 μL

M13 Forward (−20) Primer 0.1 μg/μL in TE Buffer 20 μL 2 × 20 μL

M13 Reverse Primer 0.1 μg/μL in TE Buffer 20 μL 2 × 20 μL

Sequence of primers

The table below lists the sequence of the M13 Sequencing primers included in the kit.

Primer Sequence pMoles Supplied for

10 Reaction Kit pMoles Supplied

for 20 Reaction Kit

M13 Reverse 5´-CAGGAAACAGCTATGAC-3´ 385 2 × 385

M13 Forward (–20) 5´-GTAAAACGACGGCCAG-3´ 407 2 × 407


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Kit Contents and Storage, Continued

Gel purification reagents

Some gel purification reagents are contained in Box 1. These can be removed and stored at room temperature. Other gel purification reagents are stored in the plastic bag. Store this bag at room temperature (15°C to 30°C).

Item Composition Amount for 10 Reaction Kit

Amount for 20 Reaction Kit

Sodium Iodide Solution 6.6 M Sodium iodide 16 mM Sodium sulfite

5 mL 2 × 5 mL

Binding Buffer 7 M Guanidinium HCl 7 mL 2 × 7 mL

4X Final Wash 400 mM NaCl 2.5 mL 2 × 2.5 mL

TE Buffer, pH 8 10 mM Tris-HCl 1 mM EDTA, pH 8

2 mL 2 × 2 mL

S.N.A.P.™ Purification Columns

— 11 columns 2 × 11 columns

S.N.A.P.™ Collection Vials — 11 vials 2 × 11 vials

Crystal Violet 2 mg/mL 0.5 mL 2 × 0.5 mL

6X Crystal Violet Loading Buffer

30% Glycerol 20 mM EDTA 100 μg/mL Crystal Violet

250 μL 2 × 250 μL

One Shot® reagents

The table below describes the items included in the One Shot® competent cell kit. Store at −85°C to −68°C.

Item Composition Amount for 10 Reaction Kit

Amount for 20 Reaction Kit

TOP10 OR TOP10 OR Mach1™-T1R

Chemically Competent Electrocomp™ Chemically Competent

11 × 50 μL 11 × 50 μL

—

21 × 50 μL 21 × 50 μL 21 × 50 μL

pUC19 Control DNA 10 pg/μL 50 μL 2 × 50 μL

S.O.C. Medium (may be stored at 4°C or room temperature)

2% Tryptone 0.5% Yeast Extract 10 mM NaCl 2.5 mM KCl 10 mM MgCl2 10 mM MgSO4 20 mM glucose

6 mL 2 × 6 mL

Information for non-U.S. customers using Mach1™-T1R cells

The parental strain of Mach1™-T1R E. coli is the non-K-12, wild-type W strain (ATCC #9637, S. A. Waksman). Although the parental strain is generally classified as Biosafety Level 1 (BL-1), we recommend that you consult the safety department of your institution to verify the Biosafety Level.

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Introduction

Product Overview

Description of the system

TOPO® XL PCR Cloning provides a highly efficient one-step cloning strategy ("TOPO® Cloning") for the cloning of long PCR products generated by commercial enzyme mixes specifically formulated to generate long PCR products (e.g. Elongase®, see page 25 for ordering information). No ligase, or PCR primers containing specific sequences are required. Special gel purification reagents are provided to ensure efficient cloning of long, full-length PCR products.

How it works The plasmid vector (pCR-XL-TOPO®) is supplied linearized with:

• Single 3´ thymidine (T) overhangs for TA Cloning®

• Topoisomerase covalently bound to the plasmid (referred to as "activated" vector)

Most commercial enzyme mixtures designed to generate long PCR products contain mainly Taq polymerase. Taq polymerase has a nontemplate-dependent terminal transferase activity that adds a single deoxyadenosine (A) to the 3´ ends of PCR products. The linearized vector supplied in this kit has single, overhanging 3´ deoxythymidine (T) residues. This allows PCR inserts to ligate efficiently with the vector.

Topoisomerase I from Vaccinia virus binds to duplex DNA at specific sites and cleaves the phosphodiester backbone after 5′-CCCTT in one strand (Shuman, 1991). The energy from the broken phosphodiester backbone is conserved by formation of a covalent bond between the 3′ phosphate of the cleaved strand and a tyrosyl residue (Tyr-274) of topoisomerase I. The phospho-tyrosyl bond between the DNA and enzyme can subsequently be attacked by the 5′ hydroxyl of the original cleaved strand, reversing the reaction and releasing topoisomerase (Shuman, 1994). TOPO® Cloning exploits this reaction to efficiently clone PCR products (see below). The TOPO® Cloning Reaction can be transformed directly into competent cells (provided) by either electroporation or chemical means (see Recommendation, page 9).

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Product Overview, Continued

Selecting recombinants

pCR-XL-TOPO® allows direct selection of recombinants via disruption of the lethal E. coli gene, ccdB (Bernard et al., 1994). The vector contains the ccdB gene fused to the C-terminus of the LacZα fragment. Ligation of a long PCR product disrupts expression of the lacZα-ccdB gene fusion permitting growth of only positive recombinants upon transformation. Cells that contain non-recombinant vector are killed upon plating. Therefore, blue/white screening is not required.

Lastly, by gel-purifying and visualizing the PCR product on agarose gels containing crystal violet, the damaging effects of ethidium bromide in the presence of UV light are avoided leading to higher cloning efficiencies.

We recommend electroporation as the transformation method of choice. Electroporation increases transformation efficiency of large plasmids (greater than 10 kb) over chemically competent transformation..

Experimental outline

The flow chart below outlines the experimental steps necessary to clone your long PCR product.

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Methods

Produce PCR Products

Introduction This kit is specifically designed to clone long PCR products generated by enzyme mixtures such as Elongase® Enzyme Mix. The first time you use this kit, we recommend performing the control TOPO® Cloning reaction to generate a 7 kb PCR product that can be used as a marker during agarose gel electrophoresis (see page 12) and to help you evaluate your results.

We have tested this kit specifically with the Elongase® Enzyme Mix (see page 25for ordering information). Other kits for producing long PCR products are suitable. Any enzyme mixtures that you use (commercial or homemade) should contain mainly Taq polymerase (>10:1 unit ratio) to ensure the addition of 3´ A-overhangs.

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Do not add 5´ phosphates to your primers for PCR. The PCR product synthesized will not ligate into pCR-XL-TOPO®.

Materials supplied by the user

• Enzyme mixtures and buffers specially formulated for long PCR (e.g. Elongase®)

• Thermocycler

• DNA template and primers for PCR product

• Apparatus for agarose minigel electrophoresis with 8 lane or 12 lane comb

• General purpose agarose

• Ethidium bromide

• 1X TAE buffer (40 mM Tris-acetate, pH 8, 1 mM EDTA). DO NOT USE TBE. TBE will interfere with DNA isolation using sodium iodide

Produce PCR products

Set up a 50- or 100-μL PCR reaction using the guidelines below:

• Follow the manufacturer's instructions and recommendations for producing PCR products. Optimize conditions to produce your PCR product because small PCR artifacts will preferentially clone over long PCR products.

• Use the cycling parameters suitable for your primers and template. Make sure to optimize PCR conditions to produce a single, discrete PCR product.

• Use a 7–30 minute final extension to ensure that all PCR products are completely extended.

• After cycling, place the tube on ice or store at –20ºC for up to 2 weeks. Proceed to Check the PCR Product on page 11.


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Produce PCR Products, Continued

Produce the control PCR product

The control product may be produced using buffers from the Elongase®

Amplification System. Use the manual hot start described below in order to maximize production of PCR product.

If you use another commercial mixture or a homemade mixture, you may have to optimize conditions.

1. To produce the 7-kb control PCR product, set up the following PCR in a 0.7-mL PCR tube. Use 5 μL of Elongase® 5X Buffer A and 5 μL of 5X Buffer B. The final Mg2+ concentration is 1.5 mM.

PCR Buffer (Elongase®) see above

XL Control PCR Template (25 ng/μL) 1 μL

50 mM dNTPs 0.5 μL

XL Control PCR Primers (0.2 μg/μL) 1 μL

Sterile Water to a total volume of 49 μL Final Volume 49 μL

2. For a manual hot start, begin the program listed below and pause when the heat block reaches 94°C.

Step Time Temperature Cycles

Initial Denaturation 2 minutes 94°C 1X

Denature 15 seconds 94°C

Anneal 1 minute 56°C 25X

Extend 5 minutes 68°C

Final Extension 7 minutes 72°C 1X

3. Place the tube in the block for 30 seconds.

4. Mix in 1 μL of Elongase® polymerase mix, add 1 drop of mineral oil (if necessary), and continue the program.

5. When the program is complete, place the tube on ice.

Check the PCR product

Remove 5–10 μL from each PCR amplification and analyze by agarose gel electrophoresis. Be sure you have a single, discrete band of the correct size. The control PCR amplification should produce a discrete 7 kb band and greater than 1 μg total of PCR product.

It is important to generate sufficient PCR product (greater than 0.2 μg total) to allow for losses during the following purification steps. Pool and concentrate several amplification reactions by ethanol precipitation, if necessary.

Proceed to Gel-Purify PCR Products (page 12).

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Gel Purify PCR Products

Introduction We recommend gel-purifying long PCR products to increase the cloning efficiency. However, in traditional ethidium bromide agarose gel electrophoresis, exposure of DNA to UV light may damage the DNA and significantly decrease cloning efficiency. To avoid damage to long PCR products by UV light, visualize and purify PCR products by agarose gel electrophoresis using crystal violet which is supplied in the kit (Rand, 1996). The PCR product may also be visualized under normal light as a thin blue band. PCR products can be visualized while the gel is running and excised as soon as they are sufficiently resolved.

Crystal violet is listed as a possible carcinogen, but at the concentrations supplied in the kit, it does not require a Safety Data Sheet (SDS). In addition, crystal violet and sodium iodide will stain skin and fabric. Be sure to wear gloves and a laboratory coat when using these reagents.

Crystal violet is not as sensitive as ethidium bromide. We recommend that you only use this staining method for gel-purification of DNA fragments destined to be cloned. Two hundred nanograms are barely visible on an agarose gel containing crystal violet. For isolating long PCR products, we recommend that you load greater than 200 ng per lane.

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DO NOT USE loading buffers containing bromophenol blue or xylene cyanol because the dyes will react with crystal violet and the DNA bands may become distorted. Use the 6X Crystal Violet Loading Buffer supplied in the kit.

Molecular weight markers

If you perform the control PCR reaction, you can use the 7-kb control PCR fragment as a molecular weight marker. Alternatively, 4 μg of a lambda Hind III digest mixed with 6X Crystal Violet Loading Buffer will yield visible 4.4-kb, 6.5-kb, 9.4-kb, and 23-kb bands for sizing of your PCR product.

Materials supplied by the user

• Control PCR product (page 11) • Apparatus for agarose minigel electrophoresis is with 8-lane or 12-lane comb • General purpose agarose • 1X TAE buffer (40 mM Tris-acetate, pH 8.0, 1 mM EDTA) • Clean glass flask • Autoclaved water or TE buffer • Sterile 15-mL bottle to prepare 1X Final Wash (see page 13) • New razor blade • 42–50°C water bath • Microcentrifuge


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Gel Purify PCR Products, Continued

Before starting • Bring the Sodium Iodide solution, Binding Buffer, 4X Final Wash, and TE Buffer to room temperature. Mix well to re-dissolve salts before using. Store these solutions at room temperature after the first use.

• To prepare 1X Final Wash, transfer all of the 4X Final Wash solution to a sterile 15-mL bottle. Add 7.5 mL of 100% ethanol to the 4X Final Wash solution to prepare the 1X Final Wash solution. Store at room temperature.

Nuclease control It is very important to minimize the presence of nucleases to ensure the highest

cloning efficiencies. Follow the guidelines listed below. While some guidelines may not appear as rigorous as others, they are sufficient for purifying long PCR fragments. • Wear gloves at all times

• Use sterile plasticware and glassware

• Autoclave TAE to use as the running buffer

• Rinse agarose gel apparatus and comb with autoclaved water or TE buffer

• Use a new razor to excise gel slice*

• Use new plastic wrap (e.g. SARAN WRAP™) if needed

*The same razor may be used to excise different bands in the same gel if you are careful not to bring over pieces from an earlier excision.

Prepare the gel Follow the instructions below to prepare a 0.8% agarose gel. The recipe makes one

agarose gel with a volume of 50 mL.

1. Mix 0.4 g of general purpose agarose and 50 mL 1X TAE buffer in a clean glass flask.

2. Place flask in the microwave and heat until just boiling. Swirl to dissolve agarose and continue to heat in this fashion for 3 minutes to destroy nucleases.

3. Remove the flask from the microwave and allow to cool for 3 minutes.

4. Add 20–40 μL of the 2 mg/mL Crystal Violet solution to the agarose and swirl to mix. The agarose should be light to medium purple in color.

5. Rinse the gel box and comb with autoclaved water or TE buffer.

Note: Use a comb that will hold all of the PCR amplification (48 μL) in one well, if possible.

6. Pour the gel and set the comb in the gel.

7. When the gel has solidified, cover the gel with 1X TAE buffer. You do not have to add crystal violet to the running buffer. Proceed to the next section to prepare your sample.


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Load and run the gel

1. Add 8 μL of 6X Crystal Violet Loading Buffer to 40 μL of the PCR amplification and load onto the gel. Prepare the control PCR product in the same manner.

2. Run the gel at 80 volts until the crystal violet in the gel has run about a quarter of the way UP the gel (crystal violet appears to migrate toward the negative pole). You should also see the thin blue PCR product move down into the gel. If no PCR product is visible, insufficient DNA was loaded (less than 200 ng).

3. Compare your PCR product to the 7-kb control PCR fragment. If your PCR product is sufficiently resolved so that you can easily excise the fragment, turn off the power supply. Proceed directly to Excise the PCR Product, below.

Excise the PCR product

Remember to excise the control fragment as well as your band.

1. Pour off the buffer (or transfer the gel to new Saran® Wrap).

Note: Placing the gel on a fluorescent light box may help visualize the fragments.

2. Using a new razor blade, carefully excise the desired band from the gel.

Note: The razor blade may be rinsed with autoclaved water or TE prior to cutting the next band.

3. Cut up the excised piece of agarose into small chunks and transfer to a sterile 1.5-mL microcentrifuge tube.

Note: Cutting up the agarose piece reduces the time and temperature required to melt the agarose.

4. Estimate the volume of the agarose pieces (generally this is around 100 μL). Alternatively, you can weigh the gel slice and assume that 1 mg ~ 1 μL.

5. Add 2.5 times its volume of 6.6 M sodium iodide (i.e. 250 μL) and mix by shaking vigorously by hand or vortexing.

6. Incubate at 42–50°C until the agarose is completely melted (~2 minutes). Mix the solution periodically by shaking vigorously.

7. Place the tube at room temperature and add 1.5 volumes of Binding Buffer (i.e. 525 μL) and mix well. Proceed directly to Isolate the PCR Product on page 15.


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Isolate the PCR product

1. Assemble a S.N.A.P™ purification column (A) and collection vial (B) and load all of the mixture from step 7 of

Excise the PCR Product on page 14 onto the

column (875 μL).

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2. Centrifuge the column at 2000–3000 × g in a microcentrifuge for 30 seconds at room temperature.

3. Pour the liquid in the collection vial back onto the column and repeat step 2 of this procedure.

4. Repeat step 3 of this procedure one more time to bind all of the DNA to the column (i.e. load solution onto the column for a total of 3 times).

5. After the last centrifugation, discard the liquid in the collection tube.

6. Add 400 μL of 1X Final Wash to the S.N.A.P™ column and centrifuge as in step 2 of this procedure.

7. Repeat step 6 of this procedure and discard the liquid in the collection tube after the final centrifugation (800 μL).

8. Centrifuge the column again at maximum speed (>10,000 × g) for at least 1 minute to dry the column resin. Discard the collection vial.

9. Transfer the column to a new, sterile 1.5-mL microcentrifuge tube.

10. Add 40 μL of TE buffer directly to the column material and incubate for 1 minute at room temperature to let the buffer absorb into the column.

11. Centrifuge the column at maximum speed (>10,000 × g) for 1 minute to elute the DNA into the microcentrifuge tube.

12. Place the tube on ice and discard the column.

13. Assay 10 μL by ethidium bromide agarose gel electrophoresis to estimate the DNA concentration. The Concentration should be between 2–40 ng/μL. In most cases, there is no need to concentrate the DNA further.

Store the DNA at –20°C for up to one week or proceed directly to TOPO® Cloning, page 16. For the highest efficiencies, proceed directly to TOPO® Cloning.

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TOPO® Cloning

Introduction After gel-purifying your PCR product, you are ready to TOPO Clone® it into the pCR-XL-TOPO® vector and transform the recombinant vector into competent E coli. It is important to have everything you need set up and ready to use to ensure that you obtain the best possible results. We suggest that you read this section and the section entitled Transform One Shot® Competent Cells (pages 17–19) before beginning.

TOPO® Cloning reaction

1. Set up the following 5 μL TOPO® Cloning reaction in a sterile microcentrifuge tube. Remember to set up a reaction for the control PCR product. You may also wish to set up a "vector-only" reaction as a negative control (use TE buffer instead of insert).

Gel-purified long PCR product from step 12, page 15 4 μL pCR-XL-TOPO® vector 1 μL Final Volume 5 μL

2. Mix gently and incubate for at least 5 minutes at room temperature (~25°C).

Note: Extending the incubation time up to 30 minutes may increase cloning efficiency for long PCR products.

3. After incubation, add 1 μL of the 6X TOPO® Cloning Stop Solution and mix for several seconds at room temperature.

Note: Adding the Stop Solution increases the yield of transformants by an average of 2-fold for chemically competent cells and 10-fold for electrocompetent cells.

4. Briefly centrifuge the tube and place on ice.

Note: The TOPO® Cloning reaction may be stored on ice or frozen at –20°C for up to 24 hours. You may see a decrease in the transformation efficiency, but the cloning efficiency should remain high. We recommend that you proceed immediately to Transform Competent Cells on page 17.

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Transform Competent Cells

Introduction After performing the TOPO® Cloning reaction, transform your pCR-XL-TOPO®

construct into competent E. coli provided with your kit. Protocols to transform chemically competent and electrocompetent E. coli are provided in this section.

Control reaction The 7-kb control PCR product contains the ampicillin resistance gene. To

estimate cloning efficiency, you will need to prepare LB plates containing both 50 μg/mL kanamycin and 50 μg/mL ampicillin. By plating equal amounts of the transformation mix on plates containing either kanamycin (total transformants) or kanamycin and ampicillin (recombinant transformants), you will be able to estimate cloning efficiency.

Required materials

• Electroporator (for transformation of electrocompetent cells)

• Cuvettes for electroporation (for transformation of electrocompetent cells)

• LB plates containing 50 μg/mL kanamycin or Low Salt LB plates containing 25 μg/mL Zeocin™ (see page 24 for recipes)

Note: For the control reaction you will need an additional LB plate containing 50 μg/mL kanamycin and 50 μg/mL ampicillin

• 42°C water bath (for transformation of chemically competent cells)

• 37°C shaking and non-shaking incubator

• General microbiological supplies (e.g. plates, spreaders)

Prepare for transformation

For each transformation, you will need 1 vial of competent cells and 2 selective plate.

• Thaw the vial of S.O.C. medium from Box 2 and bring to room temperature.

• Warm selective plates at 37°C for 30 minutes.

• Thaw on ice 1 vial of One Shot® competent cells for each transformation.

• For electroporation, place 0.1 cm-cuvettes on ice.


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Transform Competent Cells, Continued

pUC19 transformation control

pUC19 plasmid is included to check the transformation efficiency of the One Shot® competent cells. Transform with 10 pg per 50 μL of cells using the protocols on pages 17–19 and select for transformants on LB containing 50 μg/mL ampicillin.

Type of Cells Volume to Plate Expected Efficiency

TOP10 Chemically Competent 10 μL + 20 μL S.O.C. 1 × 109 cfu/μg DNA

Mach1™-T1R Chemically Competent

10 μL + 20 μL S.O.C. 1 × 109 cfu/μg DNA

TOP10 Electrocompetent 20 μL of a 1:10 dilution >1 × 109 cfu/μg DNA

One Shot® electroporation

We recommend electroporation over chemical transformation for higher transformation efficiency and for less bias against large recombinant plasmids.

Use ONLY electrocompetent cells for electroporation to avoid arcing.

1. Add 2 μL of the TOPO® Cloning reaction to one vial of One Shot® electrocompetent cells and mix gently. Do not mix by pipetting up and down. Avoid formation of bubbles.

2. Carefully transfer cells and DNA to a chilled 0.1-cm electroporation cuvette.

Note: The volume of cells should be between 50–80 μL to reduce the chance of arcing.

3. Electroporate your samples using your own protocol and your electroporator.

Note: If you have problems arcing, see page 19.

4. Immediately add 450 μL of room temperature S.O.C. medium and mix well.

5. Transfer the solution to a 15-mL snap-cap tube (e.g. Falcon) and shake for at least 1 hour at 37°C to allow expression of the antibiotic resistance genes.

6. Spread 20–150 μL from each transformation on a prewarmed LB plate containing 50 μg/mL kanamycin or 25 μg/mL Zeocin™. For the control reaction only, spread 50 μL each on a prewarmed LB plate containing 50 μg/mL kanamycin and on an LB plate containing 50 μg/mL kanamycin and 50 μg/mL ampicillin.

7. Incubate plates overnight at 37°C.

8. An efficient TOPO® Cloning reaction produces several hundred colonies. For the control transformation plates, see Analyze the Control Transformation, page 20. To analyze clones containing your insert, see Analyze Positive Clones, page 20.


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Transform Competent Cells, Continued Adding Stop Solution in step 3 of the TOPO® Cloning Reaction on page 16 brings the final concentration of NaCl and MgCl2 in the TOPO® Cloning reaction to 50 mM and 10 mM, respectively. To prevent arcing of your samples during electroporation, the volume of cells should be between 50–80 μL (0.1-cm cuvettes), or 100–200 μL (0.2-cm cuvettes).

If you experience arcing during transformation, try one of the following suggestions: • Reduce the voltage normally used to charge your electroporator by 10%

• Reduce the pulse length by reducing the load resistance to 100 ohms

One Shot® chemical transformation

Use this protocol only with One Shot® TOP10 or Mach1™-T1R chemically competent cells.

1. Add 2 μL of the TOPO® Cloning reaction into a vial of One Shot® cells and mix gently. Do not mix by pipetting up and down.

2. Incubate on ice for 30 minutes.

3. Heat-shock the cells for 30 seconds at 42°C without shaking.

4. Immediately transfer the tubes to ice and incubate for 2 minutes.

5. Add 250 μL of room temperature S.O.C. medium.

6. Cap the tube tightly and shake the tube horizontally at 225 rpm at 37°C for 1 hour. Place on ice.

7. Spread 50–150 μL from each transformation on a prewarmed plate. For the control transformation, spread 50 μL each on a prewarmed LB plate containing 50 μg/mL kanamycin and on an LB plate containing 50 μg/mL kanamycin and 50 μg/mL ampicillin.

8. Incubate plates overnight at 37°C.

9. An efficient TOPO® Cloning reaction produces several hundred colonies. For the control transformation plates, see Analyze the Control Transformation on page 20. To analyze clones containing your insert, see Analyze Positive Clones on page 20.

Note: Larger plasmids will produce fewer colonies.

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Analyze Transformants

Analyze the control transformation

The control PCR product contains a functional ampicillin resistance gene and only recombinant colonies will grow on kanamycin and ampicillin. Dividing the total number of colonies that grow on kanamycin and ampicillin by the number of colonies that grow on the kanamycin-only plate will give you a reasonable estimate of cloning efficiency. For chemically competent cells, greater than 70% of these colonies will be ampicillin resistant; for electrocompetent cells, greater than 80% will be ampicillin resistant.

Relatively few colonies will be produced in the vector-only reaction. Most of these will be frameshift mutations that disrupt the CcdB reading frame leading to the production of viable colonies.

Analyze positive clones

1. Pick ~10 colonies and culture them overnight in LB medium containing 50 μg/mL kanamycin. Alternatively, you may use Low Salt LB medium containing 25 μg/mL Zeocin™.

Note: If you transformed One Shot® Mach1™-T1R competent E. coli, you may inoculate overnight-grown colonies and culture them for 4 hours in prewarmed LB medium containing 50 μg/mL kanamycin or Low Salt LB medium containing 25 μg/mL Zeocin™ before isolating plasmid. For optimal results, we recommend inoculating as much of a single colony as possible.

2. Isolate plasmid DNA using your method of choice. If you need ultra-pure plasmid DNA for automated or manual sequencing, we recommend the PureLink™ HQ Mini Plasmid Purification Kit (see page 25).

3. Analyze the plasmids by restriction analysis to confirm the presence and correct orientation of the insert. Use a restriction enzyme or a combination of enzymes that cut once in the vector and once in the insert.

Sequence You may sequence your construct to confirm that your gene is cloned in the

correct orientation. The M13 Forward (–20) and M13 Reverse primers are included to help you sequence your insert. Refer to the map on page 22 for sequence surrounding the TOPO TA Cloning® site. For the full sequence of pCR-XL-TOPO®, refer to www.lifetechnologies.com/support or contact Technical Support (page 26).


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http://www.lifetechnologies.com/support

Analyze Transformants, Continued

Analyze transformants by PCR

You may wish to use PCR to directly analyze positive transformants. For PCR primers, use either the M13 Forward (–20) or the M13 Reverse primer and a primer that hybridizes within your insert. If you are using this technique for the first time, we recommend performing restriction analysis in parallel. Artifacts may be obtained because of mispriming or contaminating template. The protocol is provided below for your convenience. Other protocols are suitable.

Materials Needed

PCR SuperMix High Fidelity (see page 25)

Appropriate forward and reverse PCR primers (20 μM each)

Procedure

1. For each sample, aliquot 48 μL of PCR SuperMix High Fidelity into a 0.5-mL microcentrifuge tube. Add 1 μL each of the forward and reverse PCR primer.

2. Pick 10 colonies and resuspend them individually in 50 μL of the PCR cocktail from step 1 of this procedure. Don't forget to make a patch plate to preserve the colonies for further analysis.

3. Incubate the reaction for 10 minutes at 94°C to lyse the cells and inactivate nucleases.

4. Amplify for 20–30 cycles.

5. For the final extension, incubate at 72°C for 10 minutes. Store at 4°C.

6. Visualize by agarose gel electrophoresis.

Long-term storage After identifying the correct clone, purify the colony and make a glycerol stock

for long-term storage.

1. Streak the original colony out for single colonies on LB plates containing 50 μg/mL kanamycin or 25 μg/mL Zeocin™.

2. Isolate a single colony and inoculate into 1–2 mL of LB containing 50 μg/mL kanamycin or 25 μg/mL Zeocin™.

3. Grow until culture is at mid-log phase (between 0.6–1.0 OD600).

4. Mix 0.85 mL of culture with 0.15 mL of sterile glycerol and transfer to a cryovial.

5. Store at –80°C.

21

Appendix

Map of pCR-XL-TOPO®

pCR-XL-TOPO® map

The map below shows the features of pCR-XL-TOPO® and the sequence surrounding the TOPO TA Cloning® site. Restriction sites are labeled to indicate the actual cleavage site. The arrow indicates the start of transcription for T7 polymerase. The complete sequence of the vector is available at www.lifetechnologies.com/support or by contacting Technical Support (page 26).

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22


Troubleshoot

TOPO® Cloning The table below lists some potential problems and possible solutions to help you troubleshoot the TOPO® Cloning procedure. We recommend performing the control reaction (see page 11) on parallel with your samples to help you evaluate your results.

Problem Solution

Low yield of transformants Remember to use the 6X TOPO® Cloning Stop Solution.

Incomplete extension during PCR

Be sure to include a final extension step of 7–30 minutes during PCR. Longer PCR products will need a longer extension time.

Dilute PCR product You will need >2 ng/μL PCR product. Concentrate the amount of PCR product by ethanol precipitation or produce more product and concentrate by ethanol precipitation.

PCR cloning artifacts ("false positives")

TOPO® Cloning is very efficient for small fragments (less than 100 bp) present in certain PCR reactions. It is imperative that you gel-purify your PCR product (pages 12–15).

PCR product does not contain sufficient 3´ A-overhangs even though Taq polymerase is present in the polymerase mix.

Taq polymerase is less efficient at adding a nontemplate 3´ A next to another A. Taq is most efficient at adding a nontemplate 3´ A next to a C. You may have to redesign your primers so that they contain a 5´ G instead of a 5´ T (Brownstein et al., 1996).

Using ethidium bromide agarose gel electrophoresis to gel-purify PCR product

Intercalation of ethidium bromide into the DNA and subsequent exposure to UV light will damage the DNA leading to lower cloning efficiencies. Use the crystal violet reagents in the kit to gel-purify your PCR product.

Gel purification The table below lists some potential problems and possible solutions to help you

troubleshoot the gel purification procedure. We recommend performing the control reaction (see page 11) in parallel with your samples to help you evaluate your results.

Problem Solution

Low yield of PCR product on gel

Load more PCR product. You may have to optimize PCR conditions to obtain more than 200 ng.

Low yield of PCR product from gel purification

Be sure to melt gel slices thoroughly to ensure release of PCR product.

Be sure to use TAE to prepare the crystal violet agarose gel. TBE will interfere with isolation of the PCR product with sodium iodide.

Fragment may fail to elute from the S.N.A.P™ column. Warm TE buffer to 37°C and try eluting again.

23

Recipes

LB (Luria-Bertani) medium and plates

Do not use Luria-Bertani Medium with Zeocin™. See Low Salt LB medium below.

Composition:

1.0% Tryptone 0.5% Yeast Extract 1.0% NaCl pH 7.0

1. For 1 liter, dissolve 10 g tryptone, 5 g yeast extract, and 10 g NaCl in 950 mL deionized water.

2. Adjust the pH of the solution to 7.0 with NaOH and bring the volume up to 1 liter.

3. Autoclave on liquid cycle for 20 minutes at 15 psi. Allow solution to cool to 55°C and add antibiotic (50 μg/mL of kanamycin) if needed.

4. Store at room temperature or at 4°C.

LB agar plates

1. Prepare LB medium as above, but add 15 g/L agar before autoclaving.

2. Autoclave on liquid cycle for 20 minutes at 15 psi.

3. After autoclaving, cool to ~55°C, add antibiotic (50 μg/mL of kanamycin), and pour into 10 cm plates.

4. Allow plates to harden, then invert and store at 4°C in the dark.

Low salt LB medium

Reduce the salt in LB medium if you are using Zeocin™ for selection.

Composition:

1.0% Tryptone 0.5% Yeast Extract 0.5% NaCl pH 7.5

1. For 1 liter, dissolve 10 g tryptone, 5 g yeast extract, and 5 g NaCl in 950 mL deionized water. For plates, be sure to add 15 g/L agar.

2. Adjust the pH of the solution to 7.5 with NaOH and bring the volume up to 1 liter.

3. Autoclave on liquid cycle for 20 minutes at 15 psi. Allow solution to cool to 55°C and add Zeocin™ to a final concentration of 25 μg/mL.

4. Store at room temperature or at 4°C.

24

Accessory Products

Additional products

The table below lists additional products that may be used with the TOPO® XL PCR Cloning Kits. For more information, visit www.lifetechnologies.com/support or contact Technical Support (page 26).

Product Amount Cat. no.

Elongase® Enzyme Mix 100 reactions 10480-010

500 reactions 10480-028

One Shot® TOP10 Chemically Competent E. coli

10 reactions C4040-10



One Shot® TOP10 Electrocompetent E. coli 10 reactions C4040-50


One Shot® Mach1™-T1R Chemically Competent E. coli


PureLink® HQ Mini Plasmid Purification Kit

100 reactions K2100-01

Kanamycin 5 g 11815-024

25 g 11815-032

100 mL (10 mg/mL)

15160-054

Zeocin™ 8 × 1.25 mL R250-01

50 mL R250-05

PCR SuperMix High Fidelity 100 reactions 10790-020

25


Technical Support

Obtaining support For the latest services and support information for all locations, go to www.lifetechnologies.com/support. At the website, you can: • Access worldwide telephone and fax numbers to contact Technical Support and

Sales facilities • Search through frequently asked questions (FAQs) • Submit a question directly to Technical Support ([email protected]) • Search for user documents, SDSs, vector maps and sequences, application notes,

formulations, handbooks, certificates of analysis, citations, and other product support documents

• Obtain information about customer training • Download software updates and patches

Safety Data Sheets (SDS)

Safety Data Sheets (SDSs) are available at www.lifetechnologies.com/support.

Certificate of Analysis

The Certificate of Analysis provides detailed quality control and product qualification information for each product. Certificates of Analysis are available on our website. Go to www.lifetechnologies.com/support and search for the Certificate of Analysis by product lot number, which is printed on the box.

Limited warranty Life Technologies Corporation is committed to providing our customers with high-quality goods and services. Our goal is to ensure that every customer is 100% satisfied with our products and our service. If you should have any questions or concerns about a Life Technologies product or service, contact our Technical Support Representatives. All Life Technologies products are warranted to perform according to specifications stated on the certificate of analysis. The Company will replace, free of charge, any product that does not meet those specifications. This warranty limits the Company’s liability to only the price of the product. No warranty is granted for products beyond their listed expiration date. No warranty is applicable unless all product components are stored in accordance with instructions. The Company reserves the right to select the method(s) used to analyze a product unless the Company agrees to a specified method in writing prior to acceptance of the order. LIFE TECHNOLOGIES AND/OR ITS AFFILIATE(S) DISCLAIM ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT, EXPRESSED OR IMPLIED, INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE. IN NO EVENT SHALL LIFE TECHNOLOGIES AND/OR ITS AFFILIATE(S) BE LIABLE, WHETHER IN CONTRACT, TORT, WARRANTY, OR UNDER ANY STATUTE OR ON ANY OTHER BASIS FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING BUT NOT LIMITED TO THE USE THEREOF. Life Technologies Corporation shall have no responsibility or liability for any special, incidental, indirect or consequential loss or damage whatsoever. The above limited warranty is sole and exclusive. No other warranty is made, whether expressed or implied, including any warranty of merchantability or fitness for a particular purpose.

26


mailto:[email protected]



Purchaser Notification

Limited Use Label License: Research Use Only

The purchase of this product conveys to the purchaser the limited, non-transferable right to use the purchased amount of the product only to perform internal research for the sole benefit of the purchaser. No right to resell this product or any of its components is conveyed expressly, by implication, or by estoppel. This product is for internal research purposes only and is not for use in commercial services of any kind, including, without limitation, reporting the results of purchaser’s activities for a fee or other form of consideration. For information on obtaining additional rights, please contact [email protected] or Out Licensing, Life Technologies, 5791 Van Allen Way, Carlsbad, California 92008.

Limited Use Label License No. 30: T7 Expression System

The composition and/or use of this product is licensed to Life Technologies Corporation by Brookhaven Science Associates, LLC. The T7 expression system is based on technology developed at Brookhaven National Laboratory under contract with the U.S. Department of Energy, and is the subject of patents and patent applications assigned to Brookhaven Science Associates, LLC (BSA,). By provisions of the Distribution License Agreement granted to Life Technologies covering said patents and patent applications, Life Technologies grants you a non-exclusive sub-license under patents assigned to BSA for the use of this technology, including the enclosed materials, based upon the following conditions: 1 – these materials are to be used for non-commercial research purposes only. A separate license under patents owned by BSA is required for any commercial use, including the use of these materials for research purposes or production purposes by any commercial entity. Information about commercial license may be obtained from The Office of Technology Transfer, Brookhaven National Laboratory, Bldg. 475D, P.O. Box 5000, Upton, New York 11973-5000. Phone (516) 344-7134. 2 - No materials that contain the cloned copy of the T7 gene 1, the gene for T7 RNA polymerase, may be distributed further to third parties outside of your laboratory, unless the recipient receives a copy of this sub-license and agrees to be bound by its terms. This limitation applies to strains BL21(DE3), BL21(DE3)pLysS and BL21(DE3)pLysE, CE6, BL21-SI Competent Cells and any derivatives that are made of them. You may refuse this sub-license by returning this product unused in which case Life Technologies accept return of the product with a full refund. By keeping or using this product, you agree to be bound by the terms of this license.

Limited Use Label License No. 54: ccdB-Fusion Vectors

ccdB selection technology is described in Bernard et al., "Positive Selection Vectors Using the F Plasmid ccdB Killer Gene" Gene 148 (1994) 71-74. The purchase of this product conveys to the buyer the non-transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer (whether the buyer is an academic or for-profit entity). For licensing information for use in other than research, please contact: Out Licensing, Life Technologies Corporation, 5791 Van Allen Way, Carlsbad, California 92008; Phone (760) 603-7200 or e-mail [email protected].

27



References

Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K. (1994). Current Protocols in Molecular Biology (New York: Greene Publishing Associates and Wiley-Interscience).

Bernard, P., Gabant, P., Bahassi, E. M., and Couturier, M. (1994). Positive Selection Vectors Using the F Plasmid ccdB Killer Gene. Gene 148, 71-74.

Brownstein, M. J., Carpten, J. D., and Smith, J. R. (1996). Modulation of Non-Templated Nucleotide Addition by Taq DNA Polymerase: Primer Modifications that Facilitate Genotyping. BioTechniques 20, 1004-1010.

Rand, K. N. (1996). Crystal Violet Can Be Used to Visualize DNA Bands During Gel Electrophoresis and to Improve Cloning Efficiency. Elsevier Trends Journals Technical Tips Online, T40022.

Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, Second Edition (Plainview, New York: Cold Spring Harbor Laboratory Press).

Shuman, S. (1994). Novel Approach to Molecular Cloning and Polynucleotide Synthesis Using Vaccinia DNA Topoisomerase. J. Biol. Chem. 269, 32678-32684.

©2012 Life Technologies Corporation. All rights reserved.

The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners.

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Notes

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Notes

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