Haynes Lab:Notebook/HPK-CFP insertion into Gal4EED/Luc using CRISPR/2015/05/07

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Overview

Yesterday I performed PCR on the DBN006 insert (from colonies 1 and 3) to try and add Type IIS restriction sites to the insert. Today I'll be checking the PCR product and performing a gel purification.

PCR Verification

Gel image:

File:2015-05-07 DBN006 post-PCR.TIF

1kb+ ladder, colony 1, colony 3

The expected fragment size (~2kb) is present, but so is a large smear. Going to move forward with gel purification.

Gel Purification

Bands were excised and weighed.

Colony 1 gel mass: 0.026g

Colony 3 gel mass: 0.038g

Samples were prepped using the Sigma GenElute Gel Extraction Kit, and concentrated into a final volume of 25 µL.

DNA Concentration

Sample ID Conc. (ng/µL) St Dev (ng/µL) 260/280
DBN006 1 10.39 0.011 2.30
DBN006 3 7.60 0.733 2.28

Single-Step Digestion/Ligation Reaction

NOTE: Steps performed are from the Zhang lab's CRISPR cloning protocol, following only step 2 of the single-step digestion-ligation reaction. Volumes for inserts are calculated from their concentrations post-PCR cleanup.

Reagent Volume (µL) (colonies 1,3)
backbone vector 50 ng (0.25 µL)
Insert 1 (DBN006) 4:1 ratio 167 ng (16.1, 22.0 µL)
10x FD Buffer (clear) 2.5
10mM DTT 1.25
10mM ATP 1.25
FD BsaI 1
T4 DNA ligase 0.5
M.B. H2O 0
Total 22.6, 28.5

Incubate the ligation reaction in a thermocycler:

  1. 37°C 5 min
  2. 23°C 5 min
  3. Cycle the previous two steps for 6 cycles (total run time 1h)
  4. 4°C hold until ready to proceed

Transform with 10µL into DH5-α Turbo competent E. coli. Use a negative plate with 10µL M.B. grade H2O as a control, for 4 plates total.