RobWardenWNB:M3D1

=Expression Engineering, Day 1, 4/4/07=

Decision
We are going to delete Spt8 in the context of the following genome: FY569:  "a" ura3-52 leu2d1 spt7-223 his4-917d lys2-173R2 This will create the SALSA complex!

Forward Primer

 * Landing Sequence (first 20 bases of URA3): ATGTCGAAAGCTACATATAA
 * Tm = 54 C
 * Ta = 49 C
 * GC Ratio: 30%
 * There is also a large hairpin
 * Tail Sequence: ACTAAAGGCTCAGTTTTTTTTTTTTTCTTCTTTTACGTA
 * Complete Oligo: ACTAAAGGCTCAGTTTTTTTTTTTTTCTTCTTTTACGTAATGTCGAAAGCTACATATAA
 * Tm = 78.1 C
 * GC Ratio: 27.1%
 * Retains hairpin from landing sequence

Reverse Primer

 * Landing Sequence: Top - 5'-TGCGGCCAGCAAAACTAA-3' Reverse Compliment - 5'-TTAGTTTTGCTGGCCGCA-3'
 * Tm = 52.2 C
 * Ta = 47.2
 * GC Ratio: 50%
 * Tail Sequence: Reverse Compliment: TATGATTATGATTATGGTTATGATTATTATTACAACTCA
 * Complete Oligo: TATGATTATGATTATGGTTATGATTATTATTACAACTCATTAGTTTTGCTGGCCGCA
 * Tm = 78.5 C
 * Tm = 73.5 C
 * GC Ratio: 29.8%

PCR
PCR Mixture:

Template      		1 ul pRS406 (=100 ng) Forward Primer 		1 ul (=100 pmol) Reverse Primer 		1 ul (=100 pmol) PCR Master Mix*		20 ul of 2.5X stock (see REAGENTS LIST) H2O           		27 ul

Protocol:
 * 1) Add water to a PCR tube. Add 28 ul to another tube.
 * 2) Add primers
 * 3) Add template to first tube
 * 4) Add PCR Mix and pipette up and down
 * 5) Place in thermal cycler to undergo:
 * 6) *95° 4 minutes
 * 7) *95° 1 minute
 * 8) *40° 1 minute
 * 9) *72° 3 minute
 * 10) *repeat steps 2-4 5 times
 * 11) *95° 1 minute
 * 12) *45° 1 minute
 * 13) *72° 3 minute
 * 14) *repeat steps 6-8 5 times
 * 15) *95° 1 minute
 * 16) *50° 1 minute
 * 17) *72° 3 minute
 * 18) *repeat steps 10-12 30 times
 * 19) *72° 10 minutes
 * 20) *4° forever (or until one of the teaching faculty removes the reactions and stores them in the freezer)