Amplification of the promoter region of the gene nifH of 'R. etli
Objective
The goal was to amplify the promoter region of the gene nifH of R. etli via PCR. The final result is a 246 bp segment containg the iGEM preffix, the promoter region mentioned above, and the iGEM suffix for further manipulation of the promoter region.
Material
- H20 (HPLC)
- PCR Buffer
- MgCl2
- dNTPs
- Upper Oligo (Oligo that contains the iGEM preffix + the first 25 nts of the promoter region of nifH)
- Lower Oligo (Oligo containing the last 24 nts of the promoter region of nifH + the iGEM suffix)
- Diluted R. etli chromosomal DNA
- Taq polymerase
Workflow
1. The concentration of the oligos that are used to perform the PCR has to be 5pmol / µl. The concentration of the sinthesized upper and lower oligos is:
- [preffix_nifH_up] = 171.21 pmol / µL
- [suffix_nifH_low] = 172.02 pmol / µL
In order to obtain 500 µl of the oligos in the desired concentration, I diluted the oligos as follows:
- 14.6 µL of preffix_nifH_up + 485.4 µL of H2O
- 14.53 µL of suffix_nifH_low + 485.47 µL of H2O
2. Once the oligos are in the adequate concentration I performed a 50 µl PCR reaction as follows:
| Reagent
|
Volume (µL)
|
| H2O
|
32
|
| PCR Buffer
|
5
|
| MgCl2
|
2.5
|
| dNTPs
|
2.5
|
| preffix_nifH_up
|
2.5
|
| suffix_nifH_low
|
2.5
|
| DNA
|
2
|
| Taq Pol
|
1
|
- The control was prepared using the same quantities of each reagent except that instead of adding 2 µL of DNA, I added 34 µL of H2O.
3. To asses the size of the amplified DNA fragment the PCR products were run in a 2% agarose gel at 100 V for 1 hour. I loaded 5 µL of each PCR product + 1 µL of die. The gel was later stained with ethidium bromide. The results are pictured below.
|
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