January 2013

Chin-Sang Lab Volunteer Training

Overview

Basic Procedures

PCR, Digestions, Ligation, Transformation, Plasmid Prep, Egg Prep, Worm Lysis, Gel Electrophoresis, Gel Extraction & Clean Up Kits

Worm Care

Worm Picking Tips, Chunking, Gender Identifiers, Life Stages & Identifiers, Infected Plates

Crosses

Strain Notation, Cross Notation, Extrachromosomal & Integrated Strains, Example Problems

Overview (cont’d)

Lab Maintenance

Maintaining Sterility: Racking Tips, Retrieving Microcentrifuge Tubes, Sterile Technique, Autoclaving

General: Making Solutions, Dishwashing Procedure, Bleaching Culture Tubes

Next Steps

Tutorials, Checkpoints, Resources

Basic Procedures

The Big Picture

PCR

Digestion

LigationTransformatio

n

In All Procedures:

1. Know why you are doing the task

2. There are often many ways to perform the same task

Each procedure/kit has certain differences though, so this is why #1 is so important. By knowing the destiny of your product you can better select the method with which to make it.

3. Keep accurate and detailed notes of everything you do in the lab

PCR Basics

Used to amplify segments of DNA

Eg. Want to ligate that part into another plasmid so need more of it

Eg. Testing for the presence of a mutation

Mutant may amplify differently (eg. deletion mutation) or may have different cut sites (later shown in a digestion)

PCR

There are many different PCR kits used in the lab, each with its own attributes & protocol.

It’s important that you understand WHY you are performing the PCR (eg. is it for cloning?, to test for the presence of a gene?, etc.) That will help determine which polymerase to use

Keep notes of the PCRs you run and their purpose

PCR Polymerases

HiFi Polymerases

Able to proofread (contain exonucleases) fewer errors made∴

Used in cloning to have more accurate results

HiFi polymerases used in the lab: (will say HiFi on them)

Kapa HiFi

PCR Mastermix HiFi

PCR Polymerases

LowFi Polymerases

Not able to proofread

Used in testing for the presence of a gene mutation.

LowFi Polymerases used in the lab:

Taq Polymerase (made in the lab; protocol outlined here)

AccuStart II

PCR Reactions There are 3 basic steps that every PCR reaction will have:

Denaturation

Annealing

Extension

Here, we will outline the protocol to follow for the Taq polymerase the lab makes. Check the lab computer for the protocols for different kits.

ALWAYS CHECK THE PROTOCOL FOR THE RECIPE AND THE REACTION CONDITIONS FOR THE KIT YOU ARE USING!

PCR: Taq Protocol

Recipes

To adjust final volume, change amount of 10x PCR so the amount you add is 1/10 of the final volume and adjust ddH2O accordingly

To adjust the amount of DNA added, keeping the final volume the same, only adjust the amount of ddH2O added

Plasmid Genomic DNA Worm Lysis

DNA 1ul 5ul 10ul

10xPCR Buffer 3ul 3ul 3ul

Fwd Primer 2ul 2ul 2ul

Rev Primer 2ul 2ul 2ul

dNTPs 0.5ul 0.5ul 0.5ul

ddH2O 20.75ul 16.75ul 11.75ulTaq

Polymerase 0.75ul 0.75ul 0.75ul

Total Volume

30ul 30ul 30ul

Different amounts of DNA are used depending on what is being amplified

ddH2O is adjusted to account for the changes in

DNA volume

PCR (cont’d)

PCR Machine Settings

Most are preset

Temp Duration Purpose95°C 5 min Initial

Denaturation

95°C 30 sec Denaturation

Tm 15 sec Primer Annealing

72°C (varies) Varies based on polymerase

Extension

72°C 5 min Final Extension

To increase specificity of your PCR, increase the Tm

Polymerase Extension Times

•Taq: 1 min/kb•see sheets

x~30

PCR: Controls

Controls are important when troubleshooting failed reactions and to ensure that results obtained are valid.

PCR: Controls

Definition

Test Trial: the sample you are testing

Negative Control: should not yield any bands (test for contamination)

Positive Control: sample that will amplify our expected band. May not be possible for some PCR reactions.

PCR: Positive Controls

Positive controls can also be used to rule out other errors

Eg. Use 2 working primers to amplify another gene to rule out problems with reagents

Digestion Restriction enzymes cut DNA at palindromic sites unique to each

enzyme

Cutting makes sticky ends (in most cases). Complementary sticky ends can anneal in a ligation reaction

Recipe

Performing 1 digestion:

Can find protocol on Fermentas website

Google: Fermentas Fast Digest. Click “Complete L ist of F astD igest Restr iction E nzymes“. Select Enzyme. Click “Resources” Tab. Click “(Enzyme Name) Product Information”

Inactivate your enzyme with heat or purification.

Not all restriction enzymes are FD, although the lab mostly uses FD

enzymes.

Protocol for 1 Digestion

Plasmid PCR GenomicddH2O 15ul 16ul 30ul10x FD Buffer 2ul 3ul 5ul

DNA 1ul 10ul 10ulEnzyme 1ul 1ul 5ul

Total Volume 20ul 30ul 50ul

Incubation time @ 37°C 5 min 20 min 10 min•To digest with more than 1 enzyme, subtract the

amount of additional enzyme from the amount of ddH2O you add

Digesting More Than 1 Digestion (ie. multiple trials)

MasterMix

same incubation times

ddH2O 21.7ul

10x FD Buffer 3ul

DNA5ul (variable 1ul-10ul; depends on how

bright/concentrated the bands are; someone will tell you)

Enzyme 0.3ul-1ul

Total Volume 30ul

Ligation

Allows you to join together 2 fragments with complementary sticky ends into one piece of DNA

The overhangs of the 2 sticky ends will anneal together, so the 2 pieces being joined must have been cut with the same enzyme (or 2 enzymes that make complementary sticky ends) to create complementary overhangs

Different kits, with different attributes, can be used to obtain the same result

Fast Ligation & Overnight Ligation

same recipe proportions

different buffer & incubation times

Ligation

Recipe

5ul vector (Can be variable depending on concentration)

10ul insert (Can be variable depending on concentration)

4ul buffer (Fast Ligation: pre-aliquotted in “Ligation Buffers” box in the -20°C beside computer; Slow Ligase: in -20°C )

1ul T4 Ligase

Incubation Time

Fast: on bench (room temp), 15 min

Overnight: 15°C, overnight

Gibson Assembly

See NEBuilder for Gibson Assembly

Protocols:

http://nebuilder.neb.com

5’ 3’

5’ 3’

5’ 3’Pol +ligasePol +ligase

5’Pol +ligasePol +ligase

Chin-Sang LabGibson Assembly followed by PCR with outside primers

5’ 3’

5’Use this Gibslon assembly product as your PCR template with outside primersNote that the 5’ ends are “chewed” back. Normally this would be repaired with the vector backbone in a standard Gibson assembly reaction.

Two PCR fragments with overlapping sequences

Exonuclease chews back 5’ ends

DNA Pol fills in 3’ ends and Ligase joins

3’

Add some forward and reverse primer here?Say after 25 min? Will help fill in ends?Maybe inactivate Exonuclease first?

5’ 3’

5’Use this Gibson assembly product as your PCR template with outside primers

Forward primer

Reverse primer

5’

3’

5’Forward primerReverse primer

5’

3’

5’Forward primerReverse primer

5’5’

Forward primer

Reverse primer

The newly extended primer can serve as templates for future rounds of amplification of full length product

Primers extend

Denature

Heat Shock Transformation

1. Add 11ul of the ligation mixture to competent cells.

Competent cells are on the top shelf, right hand side of the -80°C in the hallway in a pitcher.

Cells must be kept cool for the “shock” to work. To do this, keep competent cells ①on ice at all times (unless otherwise indicated in procedure), allow the cells to ②thaw on ice rather than in your hand (gradual thawing ensures the cells aren’t harmed), use the cells immediately after removing them from the freezer.③

2. Keep mixture on ice for 15-20 min.

3. Heat shock at 42°C for 90 sec.

4. Put on ice for 1 min.

Transformations (cont’d)

5. Add 1ml 2xTY (use sterile technique).

6. Grow on shaker in the 37°C room for 45 min. Place plate with appropriate antibiotic resistance in the 37°C.

Plates found in clear 4°C fridge in the lab.

The plasmid backbone contains the antibiotic resistance gene

There are different antibiotic resistances that a plasmid can have (Ampicillin: plates are marked with a red line, Kanamycin: plates are marked with red/green line)

Giving bacteria that take up your plasmid antibiotic resistance allows you to select for the proper colony. Bacteria without the plasmid will not replicate when exposed to the antibiotic

Transformations (cont’d)

7. Plate out 100ul on the selection plate. Spin the rest of the cells down. Aspirate to 100ul.

8. Re-suspend the pellet and plate the remaining 100ul onto the plate from the 37°C room. Spread using ~5-10 beads. Place used beads in plastic bucket beside the sink.

9. Retrieve plates and look for colonies the next day.

Do not leave plates longer than overnight. Colonies will overgrow and combine. Joined colonies may not be identical genetically; individual colonies are. Come in the next day to remove the plate or ask someone in the lab to remove it for you.

Liquid Culture

Amplifies bacteria from selected colony in liquid media.

1. Pipette 3ml 2xTY + desired resistance into an autoclaved glass culture tube.

Growing the bacteria in liquid media that contains the antibiotic ensures that only the bacteria that keep the plasmid survive. If the challenge was removed, future generations in which the plasmid was rejected would grow alongside the bacteria with the plasmid of interest.

The antibiotic 2xTY is found in the clear 4℃ fridge.

2. Using a culture stick, pick one colony off the plate (try not to grab any agar) and swirl into media to remove bacteria from the tip.

3. Place the culture tube on the shaker in the 37°C room for ~16 hours. Do not leave it longer or it will overgrow.

Plasmid Prep/MiniPrep

Follow instructions in blue box that says “MiniPrep”

Box is on the shelf above where the worm plates are kept

Tips:

“Solution 1” is in the 4°C fridge

Diagnostic Digestion

Some undesired colonies may survive if during the ligation, the plasmid backbone closed without taking up the insert. Having a closed plasmid backbone will confer resistance for the cell that up took this plasmid (unless the insert is the resistance gene.)

A diagnostic digestion must be done to confirm that the surviving colony has the desired plasmid.

Diagnostic Digestion

2 methods:

1. Digest plasmid with same enzymes used to insert the insert (not ideal if the plasmid originally had an insert of a similar size).

2. Find a cut site unique to the insert.

How to make a composite part

1. Digest the plasmid and the insert with the same enzymes

2. Run both on a gel

3. Gel extract the desired bands (if needed)

4. Ligate the parts together

5. Transformation using the ligation product

6. Liquid culture

7. Miniprep

8. Diagnostic Digestion

Overview: How to Make a Composite Part

Example 1: Ligating insert into a multiple cloning site (MCS)

Diagnostic Digestion for Ex. 1

Because the section cut out of the original plasmid is only a few bp, digesting with the same 2 enzymes (EcoRI and KpnI) is a sufficient diagnostic.

Gel should reveal 2 bands, one should be the length of the plasmid backbone and the other should be the length of the insert

Overview: How to Make a Composite Part

Example 2: Switching one insert for another

Diagnostic Digestion for Ex. 2

GFP and RFP are approximately the same length. Re-digesting the ligated plasmid with the same 2 enzymes (Hindi III and Sma I) will not tell us anything. A plasmid that was initially undigested, a digested plasmid that RFP re-inserted into, and a correct plasmid that uptook the GFP will all appear the same on a gel.

To ensure you have the right plasmid, find a cut site unique to GFP.

Tip for Diagnostic Digestion

Don’t run your entire digestion on the gel. Save at least half of it in the -20°C in case something goes wrong with the gel. That way, you can quickly run a new gel instead of re-digesting your product.

Deactivating Enzymes

Check Fermentas protocol (same protocol used in digestion)

Some enzymes can be heat deactivated, others must be cleaned using the kit

2 general methods

1. Use the Enzymatic Clean-Up kit in the Blue Boxes

2. Heat the reaction in the PCR machines

Gel Electrophoresis

Separates bands of DNA based on their size

Altering the % agarose content of the gel and the voltage allow you to improve separation of the bands

**Always wear gloves when handling Ethidium Bromide (EtBr) and the gels after EtBr has been added. Only open EtBr in the fume hood. Dispose of EtBr pipette tips in the designated “EtBr Waste” bin.**

EtBr is found on the door of the fridge beside the computer

*The gel hardens quickly: once you pour the liquid gel in the casting tray put water in the beaker immediately so the remanence doesn’t solidify*

Gel Electrophoresis Protocol

Don’t run gel as long if using 2 rows of wells

If band is large, run longer. If band is small, run shorter.

Small Gel Large Gel

1x TBE 50 ml 100mlAgarose-Biotech

GradeDepends on %.

Usual 0.5 g (1%) Depends on %. Usual 1 g (1%)

EtBr 3ul 6ul

Voltage 100V 120V

Duration 60 min 60-75 min

Microwave 1 min & cool in fume hood

for ~2 min

Pour into gel casting tray & cool in fridge for at least 20 min

Gel Electrophoresis Tips

Variations

If separating bands of very similar size (eg. 200bp & 220 bp):

Increase gel %

Decrease voltage. Increases resolution. Must increase running time.

Tip

When mixing loading dye with DNA on Parafilm, place DNA on side of Parafilm previously covered with paper.

Gel Extraction

Allows you to remove a band from a gel (eg. when you are performing a ligation.)

To excise the band:

Bring the following to Ken Ko’s work room

UV glasses: in the drawer beside the sink

Razor: in the drawer beside the glasses & key

Microcentrifuge tubes: # bands excised=# tubes

Ladder Ruler

Gel Extraction (cont’d)

Place the gel with the gel tray on the UV box.

Put the glasses on, and turn the UV box on.

Do not look at the UV box without the glasses on

Keep the UV box on for as little as possible, because the UV light will damage the DNA

With the razor blade cut out your band of interest getting as little excess gel as possible.

Place the gel fragment in a microcentrifuge tube.

Ensure the UV box is turned off when you leave Walker’s lab

Gel Extraction (cont’d)

Removing the DNA from the gel

Follow instructions in blue box labelled “Gel Extraction”

Tips:

1. To measure weight of extracted gel, use scale in Bendena lab. Tare the scale using an empty microcentrifuge tube before weighing your sample.

2. To melt the gel place it in the 42℃ water bath for a longer time

Worm Lysis

Exposes DNA inside worms

Important for testing if worms are of a certain genotype

Followed by a PCR

Preparation:

Pour thermos of liquid N2

Have PicoFuge (mini centrifuge) beside the microscope where you are sitting

Gather enough PCR tubes for your samples & positive and negative controls

Determine which strain will be your positive control

Worm Lysis (cont’d) Protocol:

1. Each lysis sample contains 10ul. Mix 95% 1xPCR (if 10XPCR, dilute), 5% Proteinase K

2. Aliquot 10ul in the lid of each small PCR tube.

3. Pick the desired amount of worms.

4. Spin 2 at a time in PicoFuge.

5. Put tubes in liquid N2 immediately and leave them in there for a minimum of 10 min (longer is fine.)

6. Using the sieve beside the sink, pour the liquid N2 back into the large canister and catch the PCR tubes in the sieve.

7. Put the PCR tubes in the PCR machine under the “Worm Lysis” setting.

8. Include positive and negative controls. (Eg. +: lysis of strain with mutation of interest, -: lysis buffer)

Embryo Prep

Bleaching a plate of mixed stage worms kills all worms leaving only the embryos

Embryos left without food will arrest at L1

This protocol ensures all worms develop in synchrony by arresting all embryos in L1 before allowing them to proceed in their life cycle

Bleach Solution:

1mL 100% bleach

1mL 10M NaOH

8mL ddH2O

Embryo Prep (cont’d)

1. Add 1mL M9 to wash the plate.

2. Pipette M9 into microcentrifuge tube

3. Spin down in Picofuge

4. Aspirate

5. Add 1mL bleach solution

6. Turn tube for 90 sec to lyse worms (look at tube under microscope to see bodies dissolving)

7. Spin down

8. Aspirate

9. Add 1L bleach solution

Embryo Prep (cont’d)

10. Turn tubes for < 1 min (until clear)

11. Centrifuge at 8,000 rpm for 1 min

* Do not disturb the pellet-handle tube carefully*

12. Aspirate

*Do not aspirate the embryos*

13. Add 1mL M9

14. Repeat steps11-13 3-5 times

Check under microscope for embryos

Embryo Prep (cont’d)

15. Add 1mL M9

16. Put on shaker in the 20℃ incubator

17. Retrieve worms after 15 hours at the earliest

Worm Care

C. elegans Basics

Worms come in 3 genders:

Male , female

Hermaphrodite ⚥

It takes them about 3 days to become adults from the onset of embryogenesis

15°C: 5.5 days; 20°C: 3.5 days; 25°C: 2.5 days

Life span: 2-3 weeks (*If worms go into dauer they can remain there for ~3 months before proceeding onto L4*)

C. elegans Life Cycle

C. elegans can be grown from 16-25°C. Worms @ 25°C grow 2.1x faster than at 16°C. Worms @ 20°C grow 1.3x faster than worms at 16°C. Above stage

durations are at 22°C.

Life Stage Identification

Embryo

Laid EmbryoEmbryos in

Utero

Dauer

Non-aging state occurring after L1 due to unfavourable conditions

Subsequent life span is unaffected by dauer stage

Trigger:

Overcrowded

Starving

In temperatures too high (27°C)

Identification:

Long and skinny

Feeding ceases

Dauer (cont’d)

Behaviour:

On plate: lie motionless. Once disturbed, move rapidly.

Nictation: dauer-specific behaviour

the larva stands on its tail, waving its head in the air

Recovery:

occurs once favourable conditions are restored

within 1 hr of accessing food, the worm leaves dauer

QuickTime™ and aJVT/AVC Coding decompressorare needed to see this picture.

L4

L4 hermaphrodites are used in crosses

If they get too old they are able to fertilize themselves and are no longer useful in a cross

Gender Differences

Hermaphrodites

99.9% of the total population

Longer & fatter

Tail: Long and tapered

Sex chromosomes: XX

Males

0.1% of the total population

Slim and short

Tail: Fan extends from side

Sex chromosomes: X0

Making Males

wt males

Males are made by joining one gamete that contains an X chromosome and one that lacks sex chromosomes (0)

Gametes lacking sex chromosomes occur because of a nondisjunction event during gametogenesis

Offspring produced as a result of a mating event ( x ♂; as ⚥opposed to selfing) have a higher incidence of males ⚥because male sperm are 50:50 X:0

A line of non-mutant males can be maintained by taking 1 naturally occurring male and repeatedly crossing it into a N2 line

him mutants (High Incidence of Males)

him mutants (eg. him-5) interfere with the stable pairing of X chromosomes during meiosis, resulting in a higher frequency of nondisjunction events

him homozygous hermaphrodites will self to produce males at a 30% incidence (vs. 0.05% for N2 worms)

Identifying Males

Appearance

Smaller & skinnier than ⚥

Have a “fan” that extends from the tail

♂

Fan

Tail & Size Comparison: ♂ vs. ⚥

Fan

♂

⚥

Developing Fan

When performing crosses males should be picked before they’ve reached full maturity

An easy way to tell how mature a male is by his fan

Adult

L4

L3More severe

taper than the gradual

thinning in the ⚥

L4

L3

Adult

♂ ⚥

Male Tail: L3 & L4

Identifying Males

Behaviour

Males can often be found rubbing against hermaphrodites

Transferring Worms

When transferring worms keep the time that the plates are lidless to a minimum. This helps reduce the chances of the plate becoming contaminated.

Chunking

Used to move many worms (ie. hundreds) at once

Especially useful if plate is hard to pick from (ie. starved)

Only used to maintain homozygous lines (not heterozygous)

1. Remove sterilized spatula from EtOH & flame it

2. Cut through the agar of a populated plate using the spatula.

3. Lift the piece of agar onto the spatula and place it worm-side down onto a seeded plate. The worms will crawl out from under the chunk and onto the bacterial lawn on the new plate.

Worm Picking

Transfer one or a few worms at a time using a thin metal wire

2 Methods to Pick Worms:

Always flame pick before transferring a worm

1. Scooping the worm: Lower the tip of the wire and gently swipe the tip at the side of the worm and lift up.

2. Touch end of pick on OP50. Gently touch the back of a worm. Worm will stick to the bacteria.

Worm Picking (cont’d)

Putting the Worms Down:

Refocus the microscope on the edge of the bacterial lawn

Gently touch the surface of the agar and hold the pick there

The worm will crawl off towards the bacteria. For this reason it is best to place your pick very close to the lawn.

Worm Picking (cont’d)

Don’t leave the worm on the pick for too long or they will dry up & die

PRACTICE!

You will all be given your own plate of worms to maintain so you can practice picking. If you aren’t doing anything during your shift, practice picking until you have mastered it.

Contaminated Plates

Plates may become contaminated with mold, yeast, mites, or other bacteria

Cleaning the Stock

Chunk worms from an area away from the site of contamination onto a new plate. Place the chunk on the edge of the lawn.

Wait for the worms to crawl across the lawn before picking them onto another fresh plate.

Monitor the plate over the next few days to ensure that the contaminant did not also transfer

If the subsequent plate becomes contaminated repeat the procedure until the contaminant is removed

Contaminated Plates (cont’d)

Disposing of the Contaminated Plates

Most contaminated plates can be thrown directly in the autoclave bags

Wrap with Parafilm before throwing out if:

Mites on plate

Plate is badly contaminated

Crosses

Gene Notation

gene (allele)

eg. daf-2 (e979)

eg. daf-2 (e1370)

Allele Notation: The letter represented the lab that isolated the allele

Chin-Sang Lab: “qu”

Strain Notation

A strain can encompass many gene mutations, so using strain notation allows you to refer to a set of mutations in shorthand

DR 1979

Chin-Sang Lab: “IC”

•Lab that made the strain•All capitals

•Not italicized

•# assigned sequentially by lab as they register them

Microinjection

Makes transgenic worms

Inject DNA into the gonad

DNA is taken up into germ cell nuclei, forming extrachromosomal arrays

DNA can be delivered to many progeny in this way

∴ Microinject the mother’s gonad to see the trait in F1

Extrachromosomal Arrays

When DNA is microinjected it is not naturally incorporated into the worm’s genome; it remains extrachromosomal

Denoted by “Ex” in allele notation

Not all worms contain the DNA

eg. If the gene is GFP, some worms will not fluoresce, others will

Not all cells contain/can express the DNA. These worms are a genetic mosaic (different cells have different genotypes.)

eg. If the promoter dictates that GFP be expressed in all touch neurons, some may not fluoresce

Extrachromosomal Arrays

When crossing strains with extrachromosomal arrays:

Pick for the marker

eg. Only pick worms with GFP. Worms not expressing GFP do not have the DNA to express GFP.

The parent may or may not give the DNA to progeny

The parent NEVER gives the DNA to ALL the progeny; there will always be some that will not have the DNA.

Integrated Strain

The DNA has been integrated into a chromosome

Every worm in that strain contains the DNA

Every cell in the worm contains the DNA

Made by introducing DNA containing your gene of interest via microinjection and exposing the worms to mutagens

Mutations induce chromosome breaks

The extrachromosomal array you injected can be incorporated into the chromosome during DNA repair

C. elegans Chromosomes & Notation

5 pairs autosomes and one pair of sex chromosomes

Mutations occuring on different chromosomes are written in order

+ (I) ; + (II) ; unc-25::GFP (III) ; daf-18 (IV) ; him-5 (V) ; + (X)+ (I) ; + (II) ; daf-2 (III) ; + (IV) ; him-5 (V) ; + (X/0)

Chromosome #

•Wildtype•2 Normal

Chromosomes•wt chroms can

be omitted when writing

genotype

•Heterozygous•Both inherited chromosomes

contain a different mutation

•Heterozygous•1 inherited

chromosome=normal•1 inherited

chromosome=mutated

(I)(I)

(II)(II)

(IV)(IV)

(V)(V)

(III)(III)

++

++

unc-25::GFPdaf-

2+

daf-18

him-5

him-5

•Homozygous•Both inherited chromosomes

contain the same mutation

Sex Chromosome(s)

(X)(X/0)

++

Markers

When you are crossing in a “phenotype-less” gene you need to have a marker to show which progeny received the gene of interest

A segregation marker on the same chromosome as your gene of interest is used to follow how your gene is being segregated

The disappearance of the marker indicates the presence of the gene

Markers

eg. zdIs5 (I):

pmec-4::GFP

can be used as a segregation marker

pmec-4 is a promoter that is expressed in the touch neurons

pmec-4::GFP is a construct the lab made that fluoresces in the touch neurons. This provides a way to visualize the touch neurons.

Cross Basics

When setting up a cross pick 10 L3-L4 ♂ and 2 L4 ⚥

⚥ can be younger than L4 but NOT OLDER. past L4 start ⚥producing sperm and are able to fertilize themselves.

The 10:2 ratio ensures that the are preferentially fertilized by ⚥the males

It is imperative that you pick the correct genders.

In a cross, if even one is picked from the strain that was ⚥supposed to be the father, the predicted cross will not work.

In a selfing step, if a ♂ is picked instead of a no progeny will be ⚥produced

Set up a few trials (~3) of each cross in case something goes wrong on one of the plates.

Cross Example Background Info Desired end product:

Phenotypes:

rhIs-4: neuronal GFP (head)

oxis-12: seam-line GFP

daf-2 (III) daf-2 (III)

oxis-12 (X)

oxis-12 (X)

;

Cross ExamplerhIs-4 (III)

rhIs-4 (III)

him-5 (V)him-5 (V)

;♂ x him-5 (V)him-5 (V)

;

oxis-12 (X)

oxis-12 (X)

⚥P

rhIs-4 (III)

+ (III)

; him-5 (V)him-5 (V)

;oxis-12

(X)0

♂F1 1.0oxis-12 (X)

+ (X)

F1 are identical except ⚥for the sex chromosomes:

Generation

Probability

With him-5 there is a 30% chance of males. But, by

picking only males there is 100% chance this is the

genotype we are carrying into the next cross

daf-2 (III)daf-2 (III)

⚥

x

rhIs-4 (III)

daf-2 (III)

him-5 (V)+ (V)

oxis-12 (X)

+ (X)

;

;;

;1.0

+ (III) daf-2 (III)

him-5 (V)+ (V)

oxis-12 (X)

+ (X)

; ;The heads of other progeny

are NOT GREEN

∴ the probability of obtaining the desired genotype is 0.5. However, by picking only offspring with a green

head, there is 100% chance that this is genotype being used in the next

step.

⚥

⚥

Cross Example (cont’d)rhIs-4 (III)

daf-2 (III)

him-5 (V)+ (V)

oxis-12 (X)

+ (X)

; ;

Self

daf-2 (III) daf-2 (III)

oxis-12 (X)

oxis-12 (X)

;

rhIs-4 (III)

daf-2 (III)rhIs-4 (III)

rhIs-4 (III)

oxis-12 (X)

+ (X)

+ (X)+ (X)

Of the possible combinations for Chrom. III,

only the desired combination is NOT

GREEN.

SEGREGATION MARKER

NOT GREEN

BOTH GREEN

1.00.33

x = 0.33

⚥

Probability

Because there is only a 1/3 chance of getting the genotype we want from what we picked, we need to pick enough plates of single so ⚥that when they self, at least one will be homozygous for oxis-12.

A good number may be 12 plates of single . ⚥ Theoretically, we should see 4 plates of the desired genotype.

You need to OBTAIN RESULTS from the total number of plates you intended to or else the probability will be altered.

Eg. You pick 12 single plates and on 6 of the plates all the left ⚥the plate. Re-pick 6 single plates. If not, instead of working from a sample size of 12 with 1/3 chance of obtaining the correct genotype, you are now working from a sample size of 6 with the same odds. Although in theory you should still obtain 2 correct plates, the sample size is not big enough for this to be a likely outcome.

Cross Example (cont’d)

Of the 12 single plates, the correct plates will be those in which ALL the offspring have seam-line GFP (homozygous for oxis-12.)

CRISPR Cas9genome editing

Coming soon– still being optomised.

Just read about it here:

http://www.genetics.org/content/195/3/1181.full

http://www.genetics.org/content/195/3/635.full

Lab Maintenance

Maintaining a Sterile Environment

Autoclaving

Sterilized solutions and dry materials by creating a high P, high temp environment

Anything put in the autoclave must have autoclave tape put on it

Once in the autoclave, the tape will have black lines on it

Bottles

Autoclaved with loose caps

Flasks

Mouth covered in foil

Autoclave Settings

Dry Goods:

Normally autoclaved for 40 min with 15 min drying time

Gravity, Cycle 4

If you must combine dry and liquid things, dry good can go on a liquid cycle, HOWEVER, liquids can NEVER go on a dry cycle

The increased temperature and increased pressure quickly dissipate on a dry cycle. This causes the liquid to explode!

Autoclave Settings

Liquids:

<1L: 20 min cycle

4L: 30-50 min cycles

After autoclaving, label the bottle with its contents and the date

Removing Microcentrifuge Tubes

Reaching in with your hands can contaminate all the tubes

Instead,

Reach in with gloves; or

Shake the tubes out onto a paper towel. If more come out, tip them off the paper towel back into the stock.

Racking Pipette Tips

Rack tips with gloves on

Once pipette tip boxes are filled, close them with autoclave tape.

Sterile Technique

Prevents contaminants in the air from contaminating stock solutions

Best thing to do: minimize the time the lid is off the bottle

Flaming the bottle:

When you open a bottle, quickly touch the mouth of the bottle to the flame, followed by the lid

Do the same upon closing

Sterile Technique: 2xTY

2xTY is a bacterial growth media

Use gloves

EtOH the bench and the pipette before and after using 2xTY

Flame the bottle

General Lab Maintenance

Dishwashing Procedure

Flasks with live culture or contaminated solutions

Must be bleached before disposal in the sink

Other glassware

Wash with Alconox

Rinse 3x with hot water

Rinse 3x with distilled tap water (dH2O)

Culture Tubes

Bleaching Old Culture Tubes

Old culture tubes are stored on the top shelves until at least 1 rack has been filled

Pour the liquid into a beaker with bleach

Dispose of glass culture tube in glass container

Wash lids and place in buckets (drawers to the left of the sink) to be bleached

Culture Tubes

New Racks of Culture Tubes

Brand new culture tubes are used & topped with washed or new lids

Must be autoclaved on Dry Cycle 4 (autoclaved 40 minutes, with 15 minutes drying time)

Autoclaved tubes stored on lower shelf of the 2nd bench

Dilutions

Calculators:

http://www.endmemo.com/bio/dilution.php

http://www.restrictionmapper.org/dilutioncalc9.html

Worm Plates

Where the worms live

Make sure there are always at least ~300 plates

Plates are usually poured every two days

Worm Plates

Making Solution

3L aliquots

Made in 6L flask (liquid will expand in autoclave)

Follow recipe from book

Cover mouth of flask with tin foil and put a piece of autoclave tape on it

Autoclave on Liquid 2

Solution must be cooled to 65°C before adding in cholesterol (3ml), KPO4, CaCl2, MgCl2

Worm Plates: Preparation

Preparing the Plates

EtOH the counter where you place the plates and where you will be pouring

Remove small plates from bag and place in stacks 10 high

Have 300 plates ready

Worm Plates: Preparation

Preparing the PourBoy III

Sterilizing

Wipe the tubes with EtOH

Place the input tube in a 600mL beaker that is half filled with EtOH and warm water

Place the output tube in an empty 600mL beaker

Set the PourBoy III to Setting B and let the machine run continuously by setting the delay time to 0 sec.

Hold the tubes or they will tip the beakers over

Worm Plates: Preparation

Preparing for Clean-Up

Have a 600mL beaker half filled with water ready to microwave for when you are done pouring

Worm Plates: Preparation

To cool the flask:

Fill silver autoclave tin with cold water and place the flask inside

Swirl the flask every few minutes while cooling to ensure the agar doesn’t solidify on the edges

Once cooled, add the remaining, pre-aliquotted reagents

Worm Plates: Pouring

Set PourBoy III to either Setting B (11mL,) or Setting C (11mL, manual)

Empty the water from the autoclave tin and place the cooled flask in it, propped on an angle by a pipette tip box

Wipe the tubes with EtOH again and place the input tube at the bottom of the flask before wrapping the tin foil around the tube at the mouth of the flask to prevent contamination

Worm Plates: Pouring

Step on peddle to start pouring. Step on the peddle at any time to stop pouring

If on a manual setting, the peddle must be stepped on to start each new plate

Start from the bottom of the pile of plates, picking up the lid of the 10th plate and the other 9 plates above it

Once the plate is filled, place the stack on top and repeat for the 9th plate

If on automatic, stop the machine when changing stacks

Worm Plates: Clean-Up

With ~80 plates left place the clean-up beaker in the microwave for 5 min

Immediately once you are done pouring the plates, retrieve the beaker of hot water and start running it through the tube to remove any remnant agar

Use oven mitts when removing the water from the microwave

Fill the flask with water immediately after you are done pouring plates so the agar doesn’t solidify inside

If agar does solidify inside, remove it from the drain

Then wipe the tubes down with EtOH one last time

Worm Plates: Tips

The agar will solidify quickly in the flask, so once you start pouring, do the task from start to finish

*Do not let agar solidify in the PourBoy III*

Antibiotic Plates

Follow recipe in book for 1L portion

Antibiotics are in the freezer by the computer

Pour into larger plates (25ml/plate) using PourBoy III

Mark plates with the appropriate colour code:

Amp: red; Kan: green/red

Once the plates are dry (overnight) place them back in the clear bag that they came in

Tape the bag closed & write the antibiotic and the date the plates were poured on the tape

Store the bag of plates in the clear 4°C fridge

To Come...

Tutorials

Topics:

How to use AxioPlan

How to use Confocal Microscope

Searching the databases in the computer

If you’ve attended a tutorial it will be marked on the Volunteer Qualifications Chart so grad students can ask you to do higher level tasks as you become able to do them

Arrange a time with Tony

Take notes!

Resources for Additional Information

Lab Website:

https://qshare.queensu.ca/Users01/chinsang/www/chinsanglab/chin-sang_lab_manual.htm

WormBook

WormBase

Worm Atlas

Google!