preparing the plasmid fragment - wordpress.com...fragment lengths labeled, we colour the plasmid and...

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.


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3. Fix up the map by putting in the cut site locations andfragment lengths


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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


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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.


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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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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

173°

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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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228°

SalI 8520


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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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HindIII 100

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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

HindIII 65

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SalI 8520

HindIII 100

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pGFP

135 bp


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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


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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


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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


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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.


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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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preparing the plasmid fragment - wordpress.com...fragment lengths labeled, we colour the plasmid and...

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