Haynes Lab:Notebook/HPK-CFP insertion into Gal4EED/Luc using CRISPR/2016/02/04

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Overview

Sequence for EM7:ZeoR_v0120, submitted on Monday, has come back and the QuikChange site-directed mutagenesis worked on all three colonies picked. Moving forward with restriction digests of EM7:ZeoR and HA2 parts, followed by ligation and transformation into E. coli.

Restriction Digest

Reaction EM7:ZeoR HA2
Vector (HA2_v0120) (162 ng/µL)015
Insert (EM7:ZeoR_v0120) (134 ng/µL)150
EcoRI11
SpeI10
XbaI01
FastDigest 10x Buffer33
H2O1010
Total3030

Incubate at 37 °C for 10 minutes, then gel purify for insert (EM7:ZeoR_v0120, 463 bp) and vector (HA2_v0120, 3349 bp).

DNA Quantification

  • EM7:ZeoR (E/S digested): 7.4 ng/µL
  • HA2_v0120 (E/X digested): 23.5 ng/µL

260/280 values are a little on the high side (~2.2-2.3). Going to proceed with ligation.

Ligation

Reaction EM7:ZeoR:HA2_v0120 negative (HA2_v0120 only)
Vector (50 ng)2.12.1
Insert (13.8 ng)1.90.0
10x T4 DNA ligase buffer1.01.0
NEB T4 ligase1.01.0
H2O4.05.9

Incubate at room temperature for 10 minutes, then transform using the quick transformation method and 1 µL of ligation product. Keep the rest of the ligation product in case transformation efficiency is too low.

Ligation Results

  • Negative control: 3 colonies
  • Treatment: >200 colonies

Will pick 4 colonies, streak and start liquid cultures, plasmid prep, then verify by restriction digest.


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