Haynes Lab:Notebook/HPK-CFP insertion into Gal4EED/Luc using CRISPR/2015/12/08

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Overview

Prepping primers for Golden Gate assembly and putting together an assembly scheme.

Assembly can be viewed on Benchling, here.

PCR Set-up

Primers:

primer name sequence Tm (anneal)
pSB1A3_FWD_BsmBI CACACCACGTCTCGgatttactagtagcggccgctgcag 60.6
pSB1A3_REV_BsmBI CACACCACGTCTCGggctctagaagcggccgcgaattc 60.1
HA1_FWD_BsmBI CACACCACGTCTCCagccgctcggaggacagtactccgc 64.7
HA1_REV_BsmBI CACACCACGTCTCCaacaccggatctctagaagtcagatctagtcgg 60.4
EM7_FWD_BsmBI CACACCACGTCTCCtgttgacaattaatcatcggcatagtatatcggc 60.6
ZeoR_REV_BsmBI CACACCACGTCTCCtcagtcctgctcctcggccacga 65.4
HPK_FWD_BsmBI CACACCACGTCTCCctgactaccgggtaggggaggcgc 62.7
NLS:Stop_REV_BsmBI CACACCACGTCTCCtactcagctttacgctctagttaccttgcgct 63.6
HA2_FWD_BsmBI CACACCACGTCTCGagtaaagttaacagatcggccgcgact 60.4
HA2_REV_BsmBI CACACCACGTCTCGaatctctgtaggtagtttgtccaattatgtcacacc 61.2

Reaction setup:

Product Primer (For) Primer (Rev) Template
pSB1A3 pSB1A3_FWD_BsmBI pSB1A3_REV_BsmBI pSB1A3
HA1 HA1_FWD_BsmBI HA1_REV_BsmBI gBlock
EM7:ZeoR EM7_FWD_BsmBI ZeoR_REV_BsmBI MV8
HPK:CFP:CFP:NLS:Stop HPK_FWD_BsmBI NLS:Stop_REV_BsmBI DBN007_pSB1A3, E/S digested
HA2 HA2_FWD_BsmBI HA2_REV_BsmBI gBlock
HPK:CFP:CFP:NLS:Stop:HA2 HPK_FWD_BsmBI HA2_REV_BsmBI DBN007_pSB1A3


Prepare master mix:

Reagent Stock Volume (µL)
GC Buffer 5x 20
dNTP mix 10 mM 2
DMSO - 3
Phusion 20 U/µL 1
H2O - 74
100

For each of 5 reactions, add:

Reagent Stock Volume (µL)
PCR master - 19.1
Primer (for) 100 µM 0.2
Primer (rev) 100 µM 0.2
Template 100 ng/µL 0.5
20

PCR extension times:

Part Size (bp) Extension time (sec)
pSB1A3 2155 75
HA1 100 30
EM7-ZeoR 442 30
HPK-CFP 1966 75
HA2 99 30

Results

Gel image:

File:2015-12-09 gel.tif

HA1, EM7-ZeoR, and HA2 worked. pSB1A3 did not - probably due to incorrect template. Try again with complete template. HPK-CFP did not, probably due to off-target binding. Designed and ordered more specific primer for next attempt.

PCR Clean-up and DNA Quantification

I performed PCR clean-up on HA1, EM7-ZeoR and HA2 fractions, then quantified using the NanoDrop system.

Sample ID ng/µL 260/280
HA1_BsmBI Post PCR 49.6 1.93
EM7-ZeoR_BsmBI Post PCR 132.7 1.92
HA2_BsmBI Post PCR 72.2 1.88

Will submit the samples to the SOLS core lab for sequencing to confirm.