Haynes Lab:Notebook/HPK-CFP insertion into Gal4EED/Luc using CRISPR/2016/02/04: Difference between revisions
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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;">Today's project is...</span> | |style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;">Today's project is...</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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Incubate at 37 °C for 10 minutes, then gel purify for insert (EM7:ZeoR_v0120, 463 bp) and vector (HA2_v0120, 3349 bp). | Incubate at 37 °C for 10 minutes, then gel purify for insert (EM7:ZeoR_v0120, 463 bp) and vector (HA2_v0120, 3349 bp). | ||
==DNA Quantification== | |||
* EM7:ZeoR (E/S digested): 7.4 ng/µL | |||
* HA2_v0120 (E/X digested): 23.5 ng/µL | |||
260/280 values are a little on the high side (~2.2-2.3). Going to proceed with ligation. | |||
==Ligation== | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Reaction''' | |||
| align="center" style="background:#f0f0f0;"|'''EM7:ZeoR:HA2_v0120''' | |||
| align="center" style="background:#f0f0f0;"|'''negative (HA2_v0120 only)''' | |||
|- | |||
| Vector (50 ng)||2.1||2.1 | |||
|- | |||
| Insert (13.8 ng)||1.9||0.0 | |||
|- | |||
| 10x T4 DNA ligase buffer||1.0||1.0 | |||
|- | |||
| NEB T4 ligase||1.0||1.0 | |||
|- | |||
| H2O||4.0||5.9 | |||
|} | |||
Incubate at room temperature for 10 minutes, then transform using the quick transformation method and 1 µL of ligation product. Keep the rest of the ligation product in case transformation efficiency is too low. | |||
==Ligation Results== | |||
* Negative control: 3 colonies | |||
* Treatment: >200 colonies | |||
Will pick 4 colonies, streak and start liquid cultures, plasmid prep, then verify by restriction digest. | |||
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Latest revision as of 01:29, 27 September 2017
Today's project is... | Main project page Previous entry Next entry | |||||||||||||||||||||||||||||||||||||||||||||
OverviewSequence for EM7:ZeoR_v0120, submitted on Monday, has come back and the QuikChange site-directed mutagenesis worked on all three colonies picked. Moving forward with restriction digests of EM7:ZeoR and HA2 parts, followed by ligation and transformation into E. coli. Restriction Digest
Incubate at 37 °C for 10 minutes, then gel purify for insert (EM7:ZeoR_v0120, 463 bp) and vector (HA2_v0120, 3349 bp). DNA Quantification
260/280 values are a little on the high side (~2.2-2.3). Going to proceed with ligation. Ligation
Incubate at room temperature for 10 minutes, then transform using the quick transformation method and 1 µL of ligation product. Keep the rest of the ligation product in case transformation efficiency is too low. Ligation Results
Will pick 4 colonies, streak and start liquid cultures, plasmid prep, then verify by restriction digest. |