User:Meng Xiao He/Notebook/fall08/2008/11/22

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PCR of suicide vectors



 * ssTOPO A
 * sscco
 * sscbb
 * ssDuo
 * 100 bp ladder
 * PCR with KOentire primer pair:
 * Duo
 * cbb3
 * cco

MR-1 KO electroporation plates

 * No growth (DNA used was ssPCR + template (fair bit)):
 * original transformations
 * ones plated after (night of selection free growth → 4hrs of selection w/ Gm)

WM3064 transformants of KO vectors

 * All are indeed DAP-
 * Need to sequence to ensure whole plasmids transformed
 * Glycerol stocks made

Conjugation attempt

 * Approach 1
 * 1) Pass 4mL 10mM MgS04 with small volume of MR-1 and WM3064 strain thru. syringe filter
 * 2) Attach to new syringe w/ 1-2 mL LB DAP
 * 3) Drip some LB out, so flows thru. entire filter
 * 4) Cap with either Luer-cap from midi kit or syringe cap
 * Approach 2
 * 1) Mix o/n cultures of MR-1 and WM3064 strain
 * 2) Pellet cells
 * 3) Remove LB and add 4mL 10mM MgSO4 (tried: complete resuspension with Duo, and partial resuspension with cco and cbb)
 * 4) Pellet cells
 * 5) Replace MgSO4 w/ LB DAP

Both left in beaker in 30C incubator


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