Haynes Lab:Notebook/HPK-CFP insertion into Gal4EED/Luc using CRISPR/2015/04/27: Difference between revisions
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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;">Plasmid Verification of DBN001_pSB1A3</span> | |style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;">Plasmid Verification of DBN001_pSB1A3 & Type IIS Digestion/Ligation of DBN004_pSB1A3</span> | ||
|style="background-color: #F2F2F2" align="center"| | |style="background-color: #F2F2F2" align="center"|[[File:Report.png|frameless|link={{#sub:{{FULLPAGENAME}}|0|-11}}]][[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|[[File:Resultset_previous.png|frameless|link={{#lnpreventry:{{FULLPAGENAME}}}}]][[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]] }}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]][[File:Resultset_next.png|frameless|link={{#lnnextentry:{{FULLPAGENAME}}}}]]}} | ||
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Expected size fragments: 2132bp (backbone), 224bp (insert) | Expected size fragments: 2132bp (backbone), 224bp (insert) | ||
[[Image: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). | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Sample ID''' | |||
| align="center" style="background:#f0f0f0;"|'''Conc. (ng/µL)''' | |||
| align="center" style="background:#f0f0f0;"|'''St Dev (ng/µL)''' | |||
| align="center" style="background:#f0f0f0;"|'''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: | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Sample ID''' | |||
| align="center" style="background:#f0f0f0;"|'''Conc. (ng/µL)''' | |||
| align="center" style="background:#f0f0f0;"|'''St Dev (ng/µL)''' | |||
| align="center" style="background:#f0f0f0;"|'''260/280''' | |||
|- | |||
| DBN003 post cleanup||67.5||0.631||1.89 | |||
|} | |||
==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. Volumes for inserts are calculated from their concentrations post-PCR cleanup. | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Reagent''' | |||
| align="center" style="background:#f0f0f0;"|'''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 <i>E. coli</i>. Use a negative plate with 10µL M.B. grade H<sub>2</sub>O as a control, for 4 plates total. | |||
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Latest revision as of 00:56, 27 September 2017
Plasmid Verification of DBN001_pSB1A3 & Type IIS Digestion/Ligation of DBN004_pSB1A3 | Main project page Previous entry Next entry | ||||||||||||||||||||||||||||||||||||||||||||||||||||
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, for 4 plates total. |