User:Cassandra M Barrett/Notebook/Haynes Lab/2015/08/27

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T2A-EGFP and MV11 Ligation and Transformation

Purpose: to ligate digested vector and insert for subsequent transformation. Transform E.coli with ligated DNA

Methods:

Quantification: BB= 10ng/uL Insert= 17ng/uL

(

Using methods from Rene 5/16/15

Insert (10ng) 1uL Backbone (50ng) 5uL Roche ligation buffer 10uL Roche T4 ligase 1uL

Mix, spin Incubate at RT for 20min

Transformation:

2uL ligation mixture, H2O only control (-), pX330g control (+)- acciddentally started with only 17min left in ice incubation

Add 50uL of DH5alpha turbo cells to each tube with appropriate sample (lig mix, -control, +control) Incubate on ice for 30min Heat shock at 42C for 35sec Incubate on ice for 2min Add 750uL of SOC media (RT) to each tube Shake 37C for 1hr Plate on LB+Amp (Spin down, remove 100uL of supernatant, resuspend and plate) Incubate overnight at 37C

Results:

+ control: over grown with colonies - control: 6 colonies :( (spoke to Rene, says problem has been occurring with this strain before and problem hasn't been an issue with previous transformations) MV11+T2A-EGFP: 28 colonies

Plates wrapped and put in cold room

Next Steps: confirm with colony PCR, grow selected colonies, miniprep and positive colonies from liquid culture



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