Haynes Lab:Notebook/HPK-CFP insertion into Gal4EED/Luc using CRISPR/2016/01/04
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Switching to traditional BioBrick cloning for the insert. Order for the parts is going to be:
HA1 : HPK : CFP : CFP : NLS : Stop : polyA : EM7 : ZeoR : HA2
Need to take the following steps:
Mara has already taken care of cloning HA1 and EM7/ZeoR parts into pSB1A3.
KAH184 Restriction Digest & Gel Extraction
Restriction digest setup:
Incubate at 37 °C for 10 minutes, then run on gel at 100 V for 50 minutes along with undigested vector as control. Extract ~2000bp band and measure DNA concentration.
EDIT Just realized (while running gel) I should have digested with E/S, not X/S. Messed this one up. Will make more KAH184 plasmid. Will try performing ligation and hoping some insert makes it into the vector cut with X only.
Cut out the small band in the lane containing digested plasmid, estimated size approx. 2500bp (expected size ~2300bp). Cleaned up using Sigma gel extraction kit. Measured concentration of 6 ng/µL.