User:Cassandra M Barrett/Notebook/Open Chromatin/2015/11/11
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LCR pJET reattemped
Purpose: to verify that LCR can be successfully performed on two linear parts. Parts will be fused using LCR and ligated into a pJET vector. Ligation product will be transformed into 'E. coli' and colony PCR (and possibly sequencing) will be performed to confirm properly assembled insert (pJET has sequencing primers already designed for just outside of insertion point)
Before beginning, two samples of Gal4DB_mCh and ATF2 previously amplified on 10/4/15 and 9/18/15 were run on a gel to confirm their identities. Samples from 9/18/15 were used and diluted to 30nM.
Perform LCR on Gal4DB_mCh and ATF2 parts. Following reactions are from Ben Nyer's work on 11/9/15
Use polynucleotide kinase (PNK) to add 5' phosphate groups to DNA fragments.
Incubate at 37°C/ 30 min. Heat-inactivate PNK at 65°C/ 20 min.
Ligase Cycling Reaction
In a PCR tube, set up the following reaction.
Thermal cycler program:
Follow standard DH5alpha transformation protocol:
All five colonies from the pJET LCR plate were used to inoculate 5 separate tubes of LB+AMP broth for a plasmid miniprep. Colonies growing on the pJET H20 plate and negative control could be due to the low level of AMP resistance in the DH5 alpha turbo strain used in the lab. It is possible also that a mutation/insertion occurred in the pJET vector, shifting the kill gene out of frame.