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.


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



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.