Yeast Primers (Carolina stocks)
Gene | primers 1 | product size (bp) |
---|---|---|
Ade 1 | 5’ – GTTGGGTTTTATCTTTTGCAGTTGG - Ade1f JW1b | |
5’ – GGCGACTTGTAGTATATGTAAATCACG -Ade1r | 1040 | |
Ade 2 2 | 5’ - ATTCTCCTGCCAAACAAATAAGCAACTC - Ade2 F25 | |
5’ – ACATTCCGCCATACTGGAGGCAATAA - Ade2 R26 | 1010 | |
Ade 2 | 5’ - TTGCCAATGCCAAAGAATTTCACATC - Ade2 F29 | |
5’ - GCATTGAGCCGCCTTATATGAACTGT - Ade2 R30 | 1085 |
Master Mix
Reagent | uL |
---|---|
2x buffer (4) | 20 |
water | 12.8 |
NEB Taq | 0.2 |
Reaction Mix
Reagent | uL |
---|---|
master mix | 33 |
F primer | 2 |
R primer | 2 |
DNA | uL to get 50 ng |
(water | uL to get to 40 uL) |
Buffer 4 3 1X, pH 8.4
reagent | concentration |
---|---|
Tris-HCl | 14 mM |
KCl | 70 mM (standard taz buffer is 50 mM KCl) |
MgCl2 | 3 mM |
dNTP | 65 uM each |
Program Y-TD1 (anneal 61C -> 57C then 57C)
step | temp | time |
---|---|---|
1 | 94 | 30 s |
2 | 93 | 15 s |
3 | 61 | 20 s (-1C/cycle) |
4 | go to #2 4 times | |
5 | 93 | 15 s |
6 | 57 | 20 s |
7 | 72 | 12 s |
8 | go to #6 29 times | |
9 | 72 | 5 min |
10 | 10 | hold |
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the forward primers of each gene just miss the ATG start because of the lack of G & C bases outside the cds. ↩︎
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the 1.7 k.b. Ade2 cds was broken up into two PCR reactions overlapping by 300 bases because of its size and uncertainty over the average size of strands in our crude yeast gDNA prep, but the PCRs seemed to work well with 50 ng, and it might be possible to do the Ade2 in one PCR. ↩︎
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This is exactly the same as 1x standard taq buffer, but the [KCl] is slightly elevated to help the GC-poor primers anneal. I have just added KCl to the regular reaction before with no problem. (John Willoughby) ↩︎
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