Haynes Lab:Notebook/HPK-CFP insertion into Gal4EED/Luc using CRISPR/2015/03/26: Difference between revisions
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<b>Procedure</b> | <b>Procedure</b> | ||
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;"|'''pSB143-GFP''' | |||
|- | |||
| DNA||100ng (5uL)||600ng (3uL) | |||
|- | |||
| 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. | |||
Follow the instructions in the [http://openwetware.org/wiki/Image:Gblocks-user-guide.pdf gBlock user guide], under 'Cohesive-end restriction cloning'. | Follow the instructions in the [http://openwetware.org/wiki/Image:Gblocks-user-guide.pdf gBlock user guide], under 'Cohesive-end restriction cloning'. | ||
Revision as of 11:58, 26 March 2015
 Project name
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Entry titleToday 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:
Mix and incubate at 37°C for 15 minutes. Then heat-inactivate by incubating at 80°C for 20 minutes.
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