RobWardenWNB:M3D1
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Expression Engineering, Day 1, 4/4/07
Choosing a SAGA Subunit
Purpose | To choose a SAGA gene that when deleted will produce viable phage with an interesting phenotype. |
Notes on Genes:
Protein | Function Notes |
---|---|
Ada1 | Structural, Activator of histone acetyltransferase, Acetylation & Transciption Regulation |
Ada2 | Activator of histone acetyltransferase |
Ada3 | Glucose Regulator, histone acetyltransferase |
Gcn5 | histone acetyltransferase |
Ada5 | SAGA Integrity, Transcriptional regulator |
Spt3 | Transcription Activator (of RNAP2-dependant promoters) and Inhibitor |
Spt7 | SAGA Assembly, C-Truncation in SALSA |
Spt8 | required for SAGA-mediated inhibition at some promoters |
Spt20 | SAGA Integrity |
Sgf73 | Formation of preinitiation complex |
Sgf29 | Probably in SAGA |
Sgf11 | required for the Ubp8p association with SAGA and for H2B deubiquitylation |
Ubp8 | required for SAGA-mediated deubiquitination of histone H2B |
Sus1 | mRNA transport |
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!
Primer Design
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:
- Add water to a PCR tube. Add 28 ul to another tube.
- Add primers
- Add template to first tube
- Add PCR Mix and pipette up and down
- Place in thermal cycler to undergo:
- 95° 4 minutes
- 95° 1 minute
- 40° 1 minute
- 72° 3 minute
- repeat steps 2-4 5 times
- 95° 1 minute
- 45° 1 minute
- 72° 3 minute
- repeat steps 6-8 5 times
- 95° 1 minute
- 50° 1 minute
- 72° 3 minute
- repeat steps 10-12 30 times
- 72° 10 minutes
- 4° forever (or until one of the teaching faculty removes the reactions and stores them in the freezer)