Difference between revisions of "Part:BBa K165037:Design"
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We took a small spot of cells off of a fresh plate of W303a with a 200uL pipette tip, and ground them against the bottom of the reaction tube with the tip. This is not the time to be dainty- the idea is to break open the cells with physical force. | We took a small spot of cells off of a fresh plate of W303a with a 200uL pipette tip, and ground them against the bottom of the reaction tube with the tip. This is not the time to be dainty- the idea is to break open the cells with physical force. | ||
| − | We ran the reaction with the following program: | + | We ran the reaction with the following program:<br> |
95C- 5 min<br> | 95C- 5 min<br> | ||
30 cycles of-[<br> | 30 cycles of-[<br> | ||
Latest revision as of 19:29, 2 November 2008
TEF2 yeast constitutive promoter
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
-
Source
Amplified from the yeast genome (strain W303a) by Brown iGEM using promoter definition from:
Mumberg, Muller, and Funk: http://dx.doi.org/10.1016/0378-1119(95)00037-7
Here are the primers we used:
example: spacer + biobrick prefeix + homologous to yeast genome
TEF.fwd: GTTTCTT + attacccataaggttgtttgtgacgg + agcgttggttggtggatcaag
example: homologous to yeast genome + biobrick suffix + spacer
TEF.rev: cggtcaacgaactataattaacta + tactagtagcggccgctgcag + AAGAAAC
To do whole-cell PCR, we prepared a reaction with double the normal magnesium and no template
10 ul 5x PCR buffer
6 uL 25 mM MgCl2
1 ul 20 uM TEF.fwd primer
1 ul 20 uM TEF.rev primer
1 ul 10 mM (each) dNTP mix
.25 ul Taq polymerase
31.75 ul cartridge-purified water
We took a small spot of cells off of a fresh plate of W303a with a 200uL pipette tip, and ground them against the bottom of the reaction tube with the tip. This is not the time to be dainty- the idea is to break open the cells with physical force.
We ran the reaction with the following program:
95C- 5 min
30 cycles of-[
95C- 30s
45C- 60s
68C- 2 min
]
68C- 5min
We got a faint nonspecific product that was longer than the expected product, so gel extracted the expected product before ligating onto pSB1AK3.