Haynes Lab:Notebook/HPK-CFP insertion into Gal4EED/Luc using CRISPR/2015/03/26: Difference between revisions
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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> | |style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;">Cloning DBN001_pSB1A3 (1st attempt)</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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== | ==Overview== | ||
Today I'm going to try restriction digest of the gBlock and the vector, followed by ligation and transformation into DH5α competent E. coli cells. | |||
==Procedure== | |||
Restriction digest: | |||
In two separate tubes, digest the gBlock and the vector using the following reagents. | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Product''' | |||
| align="center" style="background:#f0f0f0;"|'''gBlock''' | |||
| align="center" style="background:#f0f0f0;"|'''pSB1A3-GFP''' | |||
|- | |||
| DNA||100ng (5µL)||600ng (3µL) | |||
|- | |||
| 10x FastDigest Buffer||3||3 | |||
|- | |||
| EcoRI||1||1 | |||
|- | |||
| SpeI||1||1 | |||
|- | |||
| Water||20||22 | |||
|- | |||
| Total||30||30 | |||
|} | |||
Mix and incubate at 37°C for 15 minutes. Then heat-inactivate by incubating at 80°C for 20 minutes. | |||
Dephosphorylate the backbone using an alkaline phosphatase. | |||
Dephosphorylation (Roche) | |||
{| {{table}} cellspacing="3" <!-- Dephos table --> | |||
|- | |||
| bgcolor=#cfcfcf | Reagent | |||
| bgcolor=#cfcfcf | Volume | |||
|- | |||
| DNA (clean digest) || up to 17 μL (340 ng) | |||
|- | |||
| 10x buffer d.p. || 2.0 | |||
|- | |||
| phosphatase || 1.0 | |||
|- | |||
| dH<sub>2</sub>O || --- | |||
|- | |||
| || 20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = 17 ng/μL | |||
|} | |||
Ligation protocol: | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Component''' | |||
| align="center" style="background:#f0f0f0;"|'''Volume''' | |||
|- | |||
| 10x Roche DNA Ligase Buffer||2 | |||
|- | |||
| Vector DNA||50ng (3 μL) | |||
|- | |||
| Insert DNA||37.5ng (12 μL) | |||
|- | |||
| water||to 20uL | |||
|- | |||
| NEB T4 DNA Ligase||1 | |||
|} | |||
Incubate at room temp. for 10 minutes, then heat inactivate at 65°C for 10 minutes. | |||
Finally, transform DH5α using the entire volume of ligated plasmid. Include one negative control (water) and one positive control (original plasmid). | |||
Latest revision as of 00:52, 27 September 2017
Cloning DBN001_pSB1A3 (1st attempt) | Main project page Previous entry Next entry | |||||||||||||||||||||||||||||||||||||||||||||
OverviewToday I'm going to try restriction digest of the gBlock and the vector, followed by ligation and transformation into DH5α competent E. coli cells. ProcedureRestriction digest: In two separate tubes, digest the gBlock and the vector using the following reagents.
Mix and incubate at 37°C for 15 minutes. Then heat-inactivate by incubating at 80°C for 20 minutes. Dephosphorylate the backbone using an alkaline phosphatase. Dephosphorylation (Roche)
Incubate at room temp. for 10 minutes, then heat inactivate at 65°C for 10 minutes.
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