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:
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.
| 
Today's project is...
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