User:Cassandra M Barrett/Notebook/Open Chromatin/2015/11/17

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pJET LCR Digest confirmation

Purpose: to confirm insertion of LCR product into pJET1.2/blunt vector. Isolated plasmid from minipreps will be digested to confirm proper insert length.

Methods:

Set up the following digest master mix with XbaI and PstI for all five plasmids, one negative control with water, and V0200 as a positive control.

Mastermix X8 (20uL per rxn volume)

  • XbaI FD 8uL
  • PstI FD 8uL
  • FD X10 Buffer 16uL
  • H20 88uL

Add 5uL of DNA to aliquoted mastermix. Use only 1uL of DNA for sample2 and + control because of high plasmid DNA concentration, add 4uL water. Incubate at 37C for 15 minutes. Run on gel to confirm at 100V, 50min.

Expected lengths:

  • V0200: 362bp, 4080bp
  • pJET1.2/blunt with no insert: 368bp, 2606pb
  • pJET1.2/blunt with proper insert: 2606bp, 2444bp

Results:

Image:pjetlcr.jpg

Both controls appear to have worked. Colonies 2-4 might be properly assembled, restriction sites within the insert most likely degraded it into bands (hadn't thought of this before...but duh that would happen...) 1 and 5 may have assembled with an oligo bridge or something. Will wait for ordered pJET1.2 primers to sequence.


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