Yeast ADE primers & pcr

Submitted by kdorfman on Mon, 08/21/2023 - 14:06

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

  1. the forward primers of each gene just miss the ATG start because of the lack of G & C bases outside the cds. ↩︎

  2. 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. ↩︎

  3. 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) ↩︎