Haynes Lab:Notebook/HPK-CFP insertion into Gal4EED/Luc using CRISPR/2015/04/27: Difference between revisions
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==Phosphorylate & Anneal Oligo Pairs== | ==Phosphorylate & Anneal Oligo Pairs== | ||
NOTE: Steps performed are from the [http://openwetware.org/wiki/Image:CRISPR_cloning_protocol_Zhang_lab.pdf Zhang lab's CRISPR cloning protocol], following <u>only</u> step 2 of the single-step digestion-ligation reaction. | NOTE: Steps performed are from the [http://openwetware.org/wiki/Image:CRISPR_cloning_protocol_Zhang_lab.pdf Zhang lab's CRISPR cloning protocol], following <u>only</u> step 2 of the single-step digestion-ligation reaction. Volumes for inserts are calculated from their concentrations post-PCR cleanup. | ||
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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 <i>E. coli</i>. Use a negative plate with 10µL M.B. grade H<sub>2</sub>O as a control. | |||
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Revision as of 18:05, 27 April 2015
Plasmid Verification of DBN001_pSB1A3 | <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page <html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html> </html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html> | ||||||||||||||||||||||||||||||||||||||||||||||||||||
OverviewI'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
Digest for 10 minutes at 37°C. Gel ElectrophoresisExpected 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 QuantificationBetter late than never... finally quantifying the amount of PCR product from amplifying the DBN003 fragment (CPK & Kozak).
Proceeding to PCR clean-up using the QIAquick PCR purification kit. DNA concentration after using the Qiagen kit:
Phosphorylate & Anneal Oligo PairsNOTE: 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.
Incubate the ligation reaction in a thermocycler:
Transform with 10µL into DH5-α Turbo competent E. coli. Use a negative plate with 10µL M.B. grade H2O as a control. |