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TUDelft/18 August 2008
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==Testing the Primers== | ==Testing the Primers== | ||
| - | A gradient PCR on the ''E. coli'' genome DNA was performed with primers coding for the three ''E. coli'' genes we want to amplify. These genes are ATOB, IDI and ISPA. PCRs were performed using a pre-made mastermix with Taq polymerase. To 25ul mastermix, 2.5ul forward and reverse primers, 1ul of template DNA (3ng/ul) and 19ul dH2O were added. The program used was as follows: | + | A gradient PCR on the ''E. coli'' genome DNA was performed with primers coding for the three ''E. coli'' genes we want to amplify. These genes are ATOB, IDI and ISPA. PCRs were performed using a pre-made mastermix with Taq polymerase. To 25ul mastermix, 2.5ul forward and reverse primers, 1ul of template DNA (3ng/ul) and 19ul dH2O were added. The program used was as follows:<br> |
| + | 1. 5' @ 95C<br> | ||
| + | 2. 1' @ 95C<br> | ||
| + | 3. 1' @ 52C (gradient of 5C divided over 12 steps)<br> | ||
| + | 4. 1' @ 72C<br> | ||
| + | 5. repeat steps 2-4 29x (total of 30 cycles)<br> | ||
| + | 6. 5' @ 72C<br> | ||
| + | 7. forever @ 4C (PCR can be stopped and stored in the fridge at any time from this point on)<br> | ||
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The PCR was performed and the products stored o/n @ 4C. | The PCR was performed and the products stored o/n @ 4C. | ||
Revision as of 13:36, 26 August 2008
Contents |
18th August
DNA extraction test
Today a gel was run with DNA soaked from the spots. To test the soaking-protocol, various steps of the protocol were performed on the DNA. Either just soaking at Tr or 20' at 42 C, and one also spinned. To test DNA concentrations before running the gel, to predict whether something should be visible, nanodrops were performed.
| Sample | ng/ul | A260/A280 |
|---|---|---|
| H2O | 0.42 | -0.24 |
| TE | 1.12 | -1.19 |
| TE + Punch, 23 C | 42.24 | 0.67 |
| TE + Punch, 42 C | 36.51 | 0.62 |
| TE + Punch, 42 C + Spinned | 44.15 | 0.61 |
This would imply that heating might not have a beneficial effect on DNA extracting, although it has to be taken into account that spot sizes are far from identical. All the 260/280 values imply low purity of the DNA, as it approaches more closely the value of protein instead of DNA. The blanc was not TE but H2O, which might have a negative effect on the ratio.
The gel run of the extracted DNA did not show any bands at all, possibly due to low quantity as nanodropped DNA was usually half of the remaining fluid. Another cause might be low quality DNA.
Testing the Primers
A gradient PCR on the E. coli genome DNA was performed with primers coding for the three E. coli genes we want to amplify. These genes are ATOB, IDI and ISPA. PCRs were performed using a pre-made mastermix with Taq polymerase. To 25ul mastermix, 2.5ul forward and reverse primers, 1ul of template DNA (3ng/ul) and 19ul dH2O were added. The program used was as follows:
1. 5' @ 95C
2. 1' @ 95C
3. 1' @ 52C (gradient of 5C 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)
The PCR was performed and the products stored o/n @ 4C.
Live stabs
Today the live stabs of a couple of biobricks and plasmids also arrived, we immediately put in o/n LB cultures of 5 ml, everything on Ampicillin.
