(r3) ProcedureGeneReplacements < Lab

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---+++ 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. 
   1 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)
   1 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). 
   1 Also make a glycerol stock of the overnight culture (200µl 80% glycerol, 1ml cells) and store in -80°C
   1 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
   1 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.)
   1 PCR purify rest of sample
   
---++++ BamH1 digestion

*You'll need to digest both vector plasmid and PCR insert. 
  
%TABLE{dataalign="center"}%
| *Reagent* | *Final concentration* | *µl* |
| Buffer 4 | 1X | 5 |
| BamH1-HF | 50 U | 2 |
| DNA | >50 ng | 2-40 |
| water | - | up to 50 |
| *total* | *-* | *50* |

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

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

---++++ Ligation

   1 Nanodrop both inserts and vector and record ng/ul
   1 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. 
   1 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* |

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
   1 Put cuvettes on ice and thaw ligation product


-- Main.LindseyWolf - 12 Oct 2011
Barrick Lab > ProtocolList > ProcedureGeneReplacements
Topic revision: r3 - 2011-10-12 - 21:50:54 - Main.LindseyWolf