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_BsmBICACACCACGTCTCGgatttactagtagcggccgctgcag60.6
pSB1A3_REV_BsmBICACACCACGTCTCGggctctagaagcggccgcgaattc60.1
HA1_FWD_BsmBICACACCACGTCTCCagccgctcggaggacagtactccgc64.7
HA1_REV_BsmBICACACCACGTCTCCaacaccggatctctagaagtcagatctagtcgg60.4
EM7_FWD_BsmBICACACCACGTCTCCtgttgacaattaatcatcggcatagtatatcggc60.6
ZeoR_REV_BsmBICACACCACGTCTCCtcagtcctgctcctcggccacga65.4
HPK_FWD_BsmBICACACCACGTCTCCctgactaccgggtaggggaggcgc62.7
NLS:Stop_REV_BsmBICACACCACGTCTCCtactcagctttacgctctagttaccttgcgct63.6
HA2_FWD_BsmBICACACCACGTCTCGagtaaagttaacagatcggccgcgact60.4
HA2_REV_BsmBICACACCACGTCTCGaatctctgtaggtagtttgtccaattatgtcacacc61.2

Reaction setup:

Product Primer (For) Primer (Rev) Template
pSB1A3pSB1A3_FWD_BsmBIpSB1A3_REV_BsmBIpSB1A3
HA1HA1_FWD_BsmBIHA1_REV_BsmBIgBlock
EM7:ZeoREM7_FWD_BsmBIZeoR_REV_BsmBIMV8
HPK:CFP:CFP:NLS:StopHPK_FWD_BsmBINLS:Stop_REV_BsmBIDBN007_pSB1A3, E/S digested
HA2HA2_FWD_BsmBIHA2_REV_BsmBIgBlock
HPK:CFP:CFP:NLS:Stop:HA2HPK_FWD_BsmBIHA2_REV_BsmBIDBN007_pSB1A3


Prepare master mix:

Reagent Stock Volume (µL)
GC Buffer5x20
dNTP mix10 mM2
DMSO-3
Phusion20 U/µL1
H2O-74
100

For each of 5 reactions, add:

Reagent Stock Volume (µL)
PCR master-19.1
Primer (for)100 µM0.2
Primer (rev)100 µM0.2
Template100 ng/µL0.5
20

PCR extension times:

Part Size (bp) Extension time (sec)
pSB1A3215575
HA110030
EM7-ZeoR44230
HPK-CFP196675
HA29930

Results

Gel image:

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 PCR49.61.93
EM7-ZeoR_BsmBI Post PCR132.71.92
HA2_BsmBI Post PCR72.21.88

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


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