Gene replacement using pKOV vector

Before beginning part 1: design primers

1. Insert should be ~1kb with approximately 500bp on either side of mutation, without disrupting neighboring genes.
2. 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)

1. Grow up overnight REL606 pKOV in LB + cam (tests camR)
2. 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).
3. Also make a glycerol stock of the overnight culture (200µl 80% glycerol, 1ml cells) and store in -80°C
4. 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)

1. Grow up appropriate stain with desired mutation (and WT for a control) in 5ml LB overnight

PCR out gene

1. PCR out desired gene with primers design as described above
2. 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.)
3. PCR purify rest of sample

BamH1 digestion

*You'll need to digest both vector plasmid and PCR insert.

1. Digest 1hr at 37°C (For the HF BamH1 you do not need to do a heat inactivation).
2. Store at -20°C or immediately run entire sample on gel for gel extraction.
3. 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.

1. Digest 15 minutes at 37°C, then 5 minutes at 65°C
2. PCR purify the AP-treated vector

Ligation

1. Nanodrop both inserts and vector and record ng/ul
2. 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.
3. 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

Your negative control should be vector without an insert (add more water to make 20µl).

Incubate at 16°C overnight

Transformation

1. Thaw electrocompetent cells on ice
2. Put cuvettes on ice and thaw ligation product

-- Main.LindseyWolf - 12 Oct 2011