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SBI4U Making a Recombinant Plasmid May 2019.notebook
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TATCGATAAGAATTCGCTTCTGCAGTCGACGCCCGGGGTAAGCTTTCTAGAGCGGCCGCCACCGCGGTGGAGCTC
Preparing the Plasmid Fragment
1. Paste into the NEB Cutter and click on "Circular" then "Submit"
2. Click "Custom Digest" and choose EcoRI, HindIII and SalI. Click "Submit" and screenshot the map. Add cut site locations and fragment lengths to the map.
SBI4U Making a Recombinant Plasmid May 2019.notebook
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3. Fix up the map by putting in the cut site locations andfragment lengths
SBI4U Making a Recombinant Plasmid May 2019.notebook
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Preparing the Insert Fragment
1. Paste the gene of interest sequence into the NEB Cutter and do a custom digest with the same enzymes you used with the plasmid to generate a similar map. Note that this sequence is linear. So just paste, submit, and custom digest.
2. Fix up the map by adding in cut site locations and fragment lengths. Also highlight the part of the fragment you want to keep.
CAGGGAATTCTAATGTCAGAATGTTCCTTCATACTGCTGTTCATACTGCTGCTCTGCTGAAGCTTCCAGTCCCAATTTAGTCGACCAC
SBI4U Making a Recombinant Plasmid May 2019.notebook
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Splice the fragments together and generate a map of your recombinant plasmid
In the ligation mix, the common sticky ends
can find each other. Then they anneal to each other and the enzyme ligase will create phosphodiester bonds between the fragments which pastes them together to form the recombinant plasmid.
SBI4U Making a Recombinant Plasmid May 2019.notebook
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Generating a Recombinant Map by Hand
Take the enzyme location and divide by the total. Multiply this by:
a) 100, if you want to figure out the percentage location of the circle.
b) 360, if you want to figure out the exact degrees to position the enzyme location.
1. EcoRI is at position 10 so
10/135 x 100 = 7.5%
10/135 x 360o = 27o
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SBI4U Making a Recombinant Plasmid May 2019.notebook
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2. HindIII from the insert fragment is next. It's 55 bp from EcoRI, so it's at position 65.
65/135 x 100% = 48%
65/135 x 360o = 173o
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HindIII 65
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SBI4U Making a Recombinant Plasmid May 2019.notebook
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3. SalI is up next. It's 20 bp further so it's at position 85
85/135 x 100% = 63%
85/135 x 360o = 227o
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HindIII 65
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SalI 8520
SBI4U Making a Recombinant Plasmid May 2019.notebook
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4. HindIII in the plasmid fragment is the last enzyme to place. It's 15 bp further so it's at position 100
100/135 x 100% = 74%
100/135 x 360o = 267o
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HindIII 65
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SBI4U Making a Recombinant Plasmid May 2019.notebook
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5. Now that the enzymes are positioned and the fragment lengths labeled, we colour the plasmid and insert fragments and put the name and total size in the middle of the map.
In this case we were using an unknown plasmid so we will initial it "p" and since the piece of paper the insert fragment was green, I'm pretending it's green fluorescent protein gene (GFP). So the plasmid will be called pGFP and has a total size of 135 bp.
EcoRI 10
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pGFP
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SBI4U Making a Recombinant Plasmid May 2019.notebook
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Generating the Recombinant Map Using Enzyme Sites and the NEB Cutter
This method requires no measurement with a protractor or percentage calculations. But it does require savy knowledge and good mouse control to cut and paste. Here's how!
Preparing the Plasmid (vector) Fragment:
1. Copy the whole sequence of the plasmid. Keep it linear for this. We can use the NEB cutter to circularize the recombinant plasmid later. Find the positions using the enzyme recognition sequences where the splicing enzymes (EcoRI at 10 and SalI at 25) will cut. Underline the stretch between them.
TATCGATAAGAATTCGCTTCTGCAGTCGACGCCCGGGGTAAGCTTTCTAGAGCGGCCGCCACCGCGGTGGAGCTC
2. Hit the space bar in the cut locations. You will need to know these exact locations by knowing the enzyme cut sites. We will replace the underlined sequence with the insert fragment during the splicing step.
TATCGATAAG AATTCGCTTCTGCAG TCGACGCCCGGGGTAAGCTTTCTAGAGCGGCCGCCACCGCGGTGGAGCTC
EcoRI SalI
SBI4U Making a Recombinant Plasmid May 2019.notebook
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Preparing the Insert Fragment
1. Copy the whole sequence of the gene of interest. Put it in bold so you will see it clearly when you get to the splicing step. Find and underline the stretches that will be discarded after digestion with the splicing enzymes. In this case, it's the end stretches that will be discarded. The insert fragment is in the middle.
CAGGGAATTCTAATGTCAGAATGTTCCTTCATACTGCTGTTCATACTGCTGCTCTGCTGAAGCTTCCAGTCCCAATTTAGTCGACCAC
2. Hit the space bar in the cut locations. CAGGG AATTCTAATGTCAGAATGTTCCTTCATACTGCTGTTCATACTGCTGCTCTGCTGAAGCTTCCAGTCCCAATTTAG TCGACCAC
EcoRI SalI
SBI4U Making a Recombinant Plasmid May 2019.notebook
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Making the Recombinant Plasmid Sequence
1. Copy the first section of plasmid sequence.
2. Copy the insert fragment and paste it beside the first section of plasmid sequence. Make sure that the spot where they join is the correct enzyme recognition sequence. In this case, EcoRI will read GAATTTC. The G will be in the plasmid and the AATTC will be in the insert fragment.TATCGATAAGAATTCTAATGTCAGAATGTTCCTTCATACTGCTGTTCATACTGCTGCTCTGCTGAAGCTTCCAGTCCCAATTTAG
3. Copy the second part of the plasmid sequence and paste it on the end of the insert fragment. Again, make sure the spot where they join is the correct enzyme site. In this case, SalI will read GTCGAC. The G will be in the insert fragment and the TCGAC will be in the plasmid sequence.
TATCGATAAGAATTCTAATGTCAGAATGTTCCTTCATACTGCTGTTCATACTGCTGCTCTGCTGAAGCTTCCAGTCCCAATTTAGTCGACGCCCGGGGTAAGCTTTCTAGAGCGGCCGCCACCGCGGTGGAGCTC
You now have the exact sequence of the recombinant plasmid.
TATCGATAAG
EcoRI
EcoRI SalI
SBI4U Making a Recombinant Plasmid May 2019.notebook
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Making the Recombinant Plasmid Map with The NEB Cutter
1. Copy the entire sequence of your recombinant plasmid map and paste it into the NEB Cutter. Click "Circular"and then click "Submit"
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2. A default map of the recombinant is drawn. Do a Custom Digest using the splicing enzymes and the fingerprint enzymes. In this case, EcoRI, HindIII and SalI.
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3. Take a screenshot of this custom map and then add in the enzyme cut site locations, fragment lengths, colour and its name.
SBI4U Making a Recombinant Plasmid May 2019.notebook
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EcoRI 10
HindIII 65
SalI 85
HindIII 100pGFP
135 bp
Proper Hand Drawing Computer Generated
As you can see, both maps are accurate and precise. So you are free to use either method when generating your recombinant plasmid map.
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