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name="ProtocolList" |
Gene replacement using pKOV vector
Before beginning part 1: design primers
- Insert should be ~1kb with approximately 500bp on either side of mutation, without disrupting neighboring genes.
- At 5' ends of both forward and reverse primers, include 1) short CG clamp (CGC), 2) BamH1 restriction site (GGATTC). So each primer should look like: 5'- CGC GGA TTC (normal PCRing primer) - 3'
Before beginning part 2: verify pKOV markers (ts, sacB, cat)
- Grow up overnight REL606 pKOV in LB + cam (tests camR)
- Dilute in saline 3x 1:100 (so 100ul in 10ml saline) and plate 100 ul on 1) LB cam @ 30 degrees, 2) LB cam @ 42 degree, and 3) LB + sucrose @ 30 degrees. Cells should grow at 30 but not at 43 or with sucrose (or at least very few colonies).
- Also make a glycerol stock of the overnight culture (200µl 80% glycerol, 1ml cells) and store in -80°C
- ALL remaining cells from the overnight culture should be spun down at 10,000xg for 10 minutes and miniprepped. You'll need your own plasmid stock for the BamH1 digestion.
Inoculate strain (day 0)
- Grow up appropriate stain with desired mutation (and WT for a control) in 5ml LB overnight
PCR out gene
- PCR out desired gene with primers design as described above
- Run gel with 5µl sample to verify PCR worked (if whole cell PCR doesn't work, gDNA extract and repeat the PCR. This should be reliable.)
- PCR purify rest of sample
BamH1 digestion
*You'll need to digest both vector plasmid and PCR insert.
| Reagent |
Final concentration |
µl |
| Buffer 4 |
1X |
5 |
| BamH1-HF |
50 U |
2 |
| DNA |
>50 ng |
2-40 |
| water |
- |
up to 50 |
| total |
- |
50 |
- Digest 1hr at 37°C (For the HF BamH1 you do not need to do a heat inactivation).
- Store at -20°C or immediately run entire sample on gel for gel extraction.
- Store purified DNA at -20°C or move on to AP treatment.
Antarctic phosphatase treatment
This is a VECTOR ONLY step! Save your insert(s) for ligation.
| Reagent |
µl |
| cut vector |
50 |
| AP buffer |
6 |
| AP enzyme |
1 |
| water |
3 |
| total |
60 |
- Digest 15 minutes at 37°C, then 5 minutes at 65°C
- PCR purify the AP-treated vector
Ligation
- Nanodrop both inserts and vector and record ng/ul
- Look up the size (in bp) of both insert and vector. You'll need both these to calculate ligation volumes because concentration is based on molarity.
- Calculate insert and vector volumes (insert will be 3x the molar concentration...see the formula below)
Insert: (30/3000)(size in bp) = # / (concentration in ng/ul) = x µl of insert
Vector: (10/3000)(size in bp) = # / (concentration in ng/ul) = y µl of vector
Ligation Reaction
| Reagent |
µl |
| ligation buffer |
2 |
| insert |
x |
| vector |
y |
| T4 ligase |
1 |
| water |
up to 20 |
| total |
20 |
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< | Your negative control should be vector without an insert (add more water to make 20µl). |
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> | Your negative control should be (cut) vector without an insert (add more water to make 20µl). |
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Incubate at 16°C overnight
Transformation |
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- Thaw electrocompetent cells on ice
- Put cuvettes on ice and thaw ligation product
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- Thaw electrocompetent cells on ice (2 tubes for controls and 1 tube for each reaction)
- Put cuvettes on ice and thaw ligation product (plus negative vector-only control)
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- Thaw uncut pKOV (this is your positive control)
- Add 1µl of each DNA condition to 40µl REL606 cells, flick to mix and pipette into cold cuvettes.
- Add 1ml room temperature SOC into the old microfuge tubes.
- Electroporate each and immediately pipette 20µl (using red tips) room temperature SOC buffer into cuvette and pipette out all liquid from cuvette into the 1.5ml microfuge tube.
- Incubate tubes shaking on their sides for 2-5 hours at 30°C to recover.
- Pipette 100µl onto LB + cam plates and grow overnight at 30°C.
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-- Main.LindseyWolf - 12 Oct 2011 |
