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
plasmid 15 5
FD 10x buffer 3 3
EcoRI 1 1
SpeI 1 0
XabaI 0 1
water 10 20

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).

File:2016-01-06 KAH184 E&S and ZeoR E&X digests.tif

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) 4.4 0
Vector DNA (50 ng) 2.8 2.8
2x Roche Rapid Ligation Buffer 8.2 5
NEB T4 Ligase 1 1
H2O 0 1.2
Total 16.4 10

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_fwd CCT TTC TAG ATA AAG TTA ACA GAT CGG CCG CGA CT 61.7
HA2_BB_rev AAG GCT GCA GCG GCC GCT ACT AGT AAT CTC TGT AGG TAG TTT GTC CAA TTA TGT CAC AC 60.0

Reaction setup:

Product Primer (For) Primer (Rev) Template
HA2_BB HA2_BB_fwd HA2_BB_rev gBlock

Prepare master mix:

Reagent Stock Volume (µL)
GC Buffer 5x 20
dNTP mix 10 mM 2
DMSO - 3
Primer (for) 100 µM 1.0
Primer (rev) 100 µM 1.0
Template 100 ng/µL 4.0
Phusion 20 U/µL 1
H2O - 68
100

PCR extension times:

Part Size (bp) Extension time (sec)
HA2 133 30