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

Plasmid Verification of DBN001_pSB1A3 & Type IIS Digestion/Ligation of DBN004_pSB1A3

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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) 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: 37°C 5 min 23°C 5 min Cycle the previous two steps for 6 cycles (total run time 1h) 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.