User:Cassandra M Barrett/Notebook/Open Chromatin/2015/09/27

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LCR Transformation Results and colony PCR

Results:

MV10 (+ control): lawn Water (- control): no growth yeah! MV10 XbaI/MBN/ no PNK: near lawn, less dense than + control MV10 XbaI/MBN/PNK: about 100-200 colonies RXN1: 7 fuzzy colonies, 1 mucoid colony RXN10: 14 fuzzy colonies RNX11: 4 fuzzy colonies, 1 mucoid colony


Colony PCR

Purpose: to verify any properly assembled constructs of MV10 with ATF2 and Gal4DB_mCh

Methods:

Create 30X of the following 1X reaction mix:

  • 2XGoTaqGreen 5uL
  • FP (CMV fwd, 10uM) 0.5uL
  • RP (DD123, 10uM) 0.5uL
  • Water 4uL

+ control: MV10 uncut - control: water

Use 1uL each of controls

Aliquot 10uL of MM per tube Pick colonies with pipette tip and incubate at RT for 10min. Swizzle and streak tip on LB+Amp plate. Incubate plate at 37C overnight. Run PCR with GoTaq protocol (60C, 30min)


Image:PCRLCR.jpg

1% agrose Gel of PCR samples run at 110V for 45min

Image:LCRGel92715.jpg

Gel shows KB+ ladders flanking both sides, colonies 1-27 in order from left to right with + then - controls

Fragments should be just under 3K, all negative...

Conclusions:

MV10 digested and blunted without PNK reanneals on itself more effectively than with PNK. What is even happening to the one that gets the PNK treatment? It should still have the 5' phosphate after cutting and blunting. Maybe PNK treatment is messing it up somehow? Is the vector without PNK treatment more desired for proper LCR? Even with a higher background noise level?

PCR didn't show any positive colonies...



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