Haynes Lab:Notebook/HPK-CFP insertion into Gal4EED/Luc using CRISPR/2015/04/30
|Ligation/Amplification of DBN002 & DBN003||Main project page|
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Going to try Type IIS Digestion/Ligation of DBN002 & DBN003, followed by PCR amplification. Also going to try conventional assembly using the BioBricks method .
Type IIS Digestion/Ligation
Incubate the ligation reaction in a thermocycler (program name DBN IIS):
--Clean-up of IIS Ligation--
Use the Qiagen kit. If possible, final volume elution of 30 µL.
Follow the method described here.
Parts to be used are KAH57 (AmCyan, AmCyan, NLS, Stop) and KAH104 (HPK, Kozak). Both are in v0120 vector. For this cloning, KAH57 will be the vector and KAH104 the insert, placed in front of the KAH57 part. Enzymes used will be E/X for KAH57 and E/S for KAH104.
During gel extraction, remove the vector from KAH57 (should be only large piece in digest) & the insert from KAH104 (540bp).
Cut out vector (0.134g) and insert (0.140g). Ran a gel extraction using Sigma GenElute Gel Extraction Kit, then measured concentration on the spectrophotometer.
Purity as measured by A260/A280 is poor, and concentration is low. Proceeding with ligation step.
50ng vector * 1/5.1 ng/µL = 10 µL purified KAH57 ng insert = 540bp/4691bp * 2 * 50ng = 11.5 ng, * 1/7.9 ng/µL = 1.5 µL purified KAH104
Incubate for 10 minutes at room temperature.
Transform DH5α-Turbo with: