User:Cassandra M Barrett/Notebook/Open Chromatin/2015/09/07

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LCR Results

Results from LCR performed on 9/6/15

LCR1 Results 9 6 15.jpg

The figure shows plates from the transformation performed with different LCR reactions from 9/6/15. LCR reaction 1, done with phosphorylated MV10, shows just over a dozen wide rippling colonies LCR reaction 2, done with unphosphorylated MV10, shows several hundred similar colonies in a lawn with low density LCR reaction 3, done with no ligase as a control, shows about 15 similarly hazy colonies The positive control with circular MV10 plasmid shows a lawn of growth, with small punctiform colonies The negative control, water only in the transformation, shows two typical E. coli colonies

Conclusions:

Both the negative and positive controls turned out as expected. Rene had said there might be very low levels of ampicillin resistance in the DH5alpha turbo stock, so the two colonies growing on the negative control should not be an issue as they haven't interfered with results previously. Results from the LCR reactions are particularly informative if they can be confirmed with a colony PCR. With the phosphorylated vector giving about as many colonies as the no-ligase control, we can at least see that phosphorylating the vector greatly increases the level of viable vectors that can make it into the bacteria. Doubtful, however, is if all of these colonies on the LCR2 plate are successfully ligated containing both inserts. Phosphorylating the vector in blunt end ligation reactions is done to prevent backbone from reannealing to itself. It is very possible that the blunt ends from the Mung Bean Nuclease digest, when left unphosphorylated, simply reannealed to create viable plasmid for conferring resistance in the bacteria.

Next step: confirm insertion of both ATF2 and Gal4DB_mCh into MV10 for colonies from both LCR1 and LCR2 reaction plates.

LCR2 plate diluted by adding 1.5mL LB to surface, rolling beads around. 200uL taken and plated, 200uL taken and added to 300uL of LB. 200uL of that dilution taken and plated. Both plates incubated at 37C overnight.