Haynes Lab:Notebook/HPK-CFP insertion into Gal4EED/Luc using CRISPR/2016/01/06

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Overview

Cloning HPK-CFP part (from KAH184) in front of EM7:ZeoR in v0120 vector. Also performing PCR on HA2 fragment to add BioBrick restriction sites.

KAH184 plasmid is at 220 ng/µL. EM7:ZeoR_v0120 plasmid is at 629 ng/µL.

Restriction Digest

Reagent KAH184 EM7:ZeoR
plasmid155
FD 10x buffer33
EcoRI11
SpeI10
XabaI01
water1020

Incubate at 37 °C for 10 minutes.

Gel Extraction

ladder - CFP - zeoR

HPK-CFP part: take the ~2200bp fragment.

EM7-ZeoR part: large fragment (~3600bp).

DNA Quantification

EM7:ZeoR_v0120 - 18 ng/µL

HPK:CFP - 17 ng/µL

NOTE: 260/280 for HPK:CFP part was measured at 4. Ran a spectrophotometric scan on the sample and there's no peak at 260 or 280 - probably very little DNA present in the sample. If transformation fails, this is most likely culprit.

Ligation

Reagent Ligation Neg. control
Insert DNA (75 ng)4.40
Vector DNA (50 ng)2.82.8
2x Roche Rapid Ligation Buffer8.25
NEB T4 Ligase11
H2O01.2
Total16.410

Incubate at RT for 10 minutes.

Transformation

Follow the traditional transformation protocol found here.

Homology Arm 2 PCR

Primers:

primer name sequence Tm (anneal)
HA2_BB_fwdCCT TTC TAG ATA AAG TTA ACA GAT CGG CCG CGA CT61.7
HA2_BB_revAAG GCT GCA GCG GCC GCT ACT AGT AAT CTC TGT AGG TAG TTT GTC CAA TTA TGT CAC AC60.0

Reaction setup:

Product Primer (For) Primer (Rev) Template
HA2_BBHA2_BB_fwdHA2_BB_revgBlock

Prepare master mix:

Reagent Stock Volume (µL)
GC Buffer5x20
dNTP mix10 mM2
DMSO-3
Primer (for)100 µM1.0
Primer (rev)100 µM1.0
Template100 ng/µL4.0
Phusion20 U/µL1
H2O-68
100

PCR extension times:

Part Size (bp) Extension time (sec)
HA213330




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