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TUDelft/20 August 2008
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==Testing the Primers (part 2)== | ==Testing the Primers (part 2)== | ||
| - | Today another PCR was set up, again using the mastermix and composition also used for the first experiment (18th of August). Only this time the ''S. cerevisiae'' genes and cDNA were used. The cDNA contained 2ng/ul and the genes are: ERG8, ERG12, ERG13, HMG2 and MVD1. PCR program (with a 6C gradient this time) is as depicted below: | + | Today another PCR was set up, again using the mastermix and composition also used for the first experiment (18th of August). Only this time the ''S. cerevisiae'' genes and cDNA were used. The cDNA contained 2ng/ul and the genes are: ERG8, ERG12, ERG13, HMG2 and MVD1. PCR program (with a 6C gradient this time) is as depicted below:<br> |
| - | 1. 5' @ 95C | + | 1. 5' @ 95C<br> |
| - | 2. 1' @ 95C | + | 2. 1' @ 95C<br> |
| - | 3. 1' @ 54C (gradient of 6C divided over 12 steps) | + | 3. 1' @ 54C (gradient of 6C divided over 12 steps)<br> |
| - | 4. 1' @ 72C | + | 4. 1' @ 72C<br> |
| - | 5. repeat steps 2-4 29x (total of 30 cycles) | + | 5. repeat steps 2-4 29x (total of 30 cycles)<br> |
| - | 6. 5' @ 72C | + | 6. 5' @ 72C<br> |
| - | 7. forever @ 4C (PCR can be stopped and stored in the fridge at any time from this point on) | + | 7. forever @ 4C (PCR can be stopped and stored in the fridge at any time from this point on)<br> |
This time, the products were put directly on gel. The result is shown below: | This time, the products were put directly on gel. The result is shown below: | ||
Revision as of 09:24, 26 August 2008
20th August
Testing the Primers (part 2)
Today another PCR was set up, again using the mastermix and composition also used for the first experiment (18th of August). Only this time the S. cerevisiae genes and cDNA were used. The cDNA contained 2ng/ul and the genes are: ERG8, ERG12, ERG13, HMG2 and MVD1. PCR program (with a 6C gradient this time) is as depicted below:
1. 5' @ 95C
2. 1' @ 95C
3. 1' @ 54C (gradient of 6C divided over 12 steps)
4. 1' @ 72C
5. repeat steps 2-4 29x (total of 30 cycles)
6. 5' @ 72C
7. forever @ 4C (PCR can be stopped and stored in the fridge at any time from this point on)
This time, the products were put directly on gel. The result is shown below:
STILL HAVE TO EDIT AND PUT IN THE PICTURE!!
Midiprep
All the plasmids from live stabs have been midiprepped. The gel showed multiple bands on pSB1A7, also the main band of this one was higher than the others. The rest looked OK. Concentrations of DNA were all between 40 - 100 ng/ul with a 260/280 of 1.8 +- 0.1.
