Haynes Lab:Notebook/HPK-CFP insertion into Gal4EED/Luc using CRISPR/2016/01/04

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Overview

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:

  • restriction digest of KAH184 (X/S) & gel extraction of insert for HPK-CFP part, followed by seq verification
  • PCR amplification of HA2, adding X & S/N/P cut sites to the part, then cloning into pSB1A3 backbone, followed by seq verification

Mara has already taken care of cloning HA1 and EM7/ZeoR parts into pSB1A3.

KAH184 Restriction Digest & Gel Extraction

Restriction digest setup:

Reagent Volume (µL)
KAH184 15
FD Buffer 10x 3
XbaI 1
SpeI 1
H2O 10
Total 30

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.


Gel:

File:2016-01-04 KAH184 X&S digest.tif

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.