User:Cassandra M Barrett/Notebook/Open Chromatin/2015/11/30

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Blunt Vector Construction

Purpose: to build a blunt version of MV10 containing a SmaI cut site instead of a XbaI cut site for LCR

Methods:

Based off of the following protocol:

https://www.openwetware.org/wiki/User:Karmella_Haynes/Notebook/BioBrick_cloning/2013/03/20

Annealing the oligos

Dilute oligos (MV10_SmaI_InsF and MV10_SmaI_InsR, both TM=63) to 100uM. Add 6uL of each to PCR tube with 2uL 10X annealing buffer and 6uL water. Heat to 95C for 5 minutes. Take out of heat block and let cool to room temperature for 1 hour, the put on ice until ready to use.

MV10 Digest and Dephosphorylation

Meanwhile, digest MV10 with XbaI fast digest (2uL MV10, 1uL XbaI, 1uL 10X FD buff, 6uL water) for 15min at 37C. Run on gel with control lanes of water and uncut MV10. Clean up appropriate band with gel clean up kit. Measure concentration ( 19ng/uL =( )

Dephosphorylate vector as follows (20uL total rxn volume):

  • MV10/XbaI (500 ng) 17 uL
  • 10X buff dp 2uL
  • Rapid Alk Phosphatase 1uL

Incubate at 37C for 10 min, then 75C for 2minutes to deactivate.

Ligation

Set up the following ligation to create the MV10_SmaI vector:

  • Annealed oligos 2uL
  • Vector 2uL
  • 10X T4 ligation buffer 1uL
  • T4 ligase 1uL
  • Water 4uL

Incubate 10 minutes at RT. Add 30uL DH5alpha turbo cells. Do the same for a water - control and an MV10 + control. Incubate 5minutes on ice. Plate on LB+Amp and incubate at 37C overnight.

Conclusions: Blunt vector assembly failed, no colonies...