Haynes Lab:Notebook/HPK-CFP insertion into Gal4EED/Luc using CRISPR/2015/04/27

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Owwnotebook icon.pngPlasmid Verification of DBN001_pSB1A3 & Type IIS Digestion/Ligation of DBN004_pSB1A3 <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
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Overview

I'll be performing a restriction digest on the DNA harvested from the DBN001_pSB1A3 ligation & transformation, then running the digestion products on a gel to check size fragments. Hopefully I'll see size fragments that correspond to a successful ligation.

Restriction Digest

Reagent Volume
DNA 600ng (2.5 - 4.7 µL)
10x FastDigest Buffer 3
EcoRI 1
SpeI 1
Water 22.5 - 20.3
Total 30

Digest for 10 minutes at 37°C.

Gel Electrophoresis

Expected size fragments: 2132bp (backbone), 224bp (insert)

2015-04-28 DBN001 pSB1A3 digest annotated.png

Colonies 1, 2, and 5 have bands of the expected sizes. Going to go ahead and perform Type IIS Restriction Digest/Ligation.

DBN003 Post-PCR DNA Quantification

Better late than never... finally quantifying the amount of PCR product from amplifying the DBN003 fragment (CPK & Kozak).

Sample ID Conc. (ng/µL) St Dev (ng/µL) 260/280
DBN003 408 17.94 1.87

Proceeding to PCR clean-up using the QIAquick PCR purification kit.

DNA concentration after using the Qiagen kit:

Sample ID Conc. (ng/µL) St Dev (ng/µL) 260/280
DBN003 post cleanup 67.5 0.631 1.89

Phosphorylate & Anneal Oligo Pairs

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, 2, 5)
✓ backbone vector 100 ng (0.5, 0.6, 0.7 µL)
✓ Insert 1 (DBN002) 2:1 ratio 94 ng (5.6 µL)
✓ Insert 2 (DBN003) 2:1 ratio 48 ng (0.7 µL)
✓ 10x FD Buffer (clear) 2
✓ 10mM DTT 1
✓ 10mM ATP 1
✓ FD BsaI 1
✓ T4 DNA ligase 0.5
✓ M.B. H2O 7.7, 7.6, 7.5
Total 20

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.