"
Team:DTU-Denmark/Notebook/11 July 2013
From 2013.igem.org
Contents |
208
Main purpose
- PCR with USER primers to combine Nir operon with pZA21
- PCR with USER primer to test the activity of the promoter AraB.
Who was in the lab
Ariadni, Henrike, Julia, Jakob, Gosia
Procedure
Combine Nir operon with pZA21
Following Mathildes Protocol for DpnI digestion of our linear pZA21 w. Uracil incerts
Mastermix for 7 PCR tubes, Using the mastermix protocol we got from Mathilde.
triplets of each of the two Nir Operon extracts that we have made yesterday, including one negative control:
- Nir 1(a, b, c)
- Nir 2(a, b, c)
Using the following PCR program:
| time | Temp C | |
|---|---|---|
| Denaturing | 2 min | 98 |
| start of loop | 10 sec | 98 |
| annealing | 30 sec | 60 |
| elongation | 9 min | 72 |
| goto start of loop 35 times | ||
| hold | 10 |
PCR for promoter test
We want to test the inducible AraBAD promoter using plasmid pZA21 without its native promoter and RFP gene. We performed 3 different PCR reactions using user primers. Each PCR reaction was performed in triplicate.
Samples are named:
- 1,2,3 -> pZA21 with no native promoter - Primers 13a and 13b, template pZA21 miniprep
- 4,5,6 -> RFP - Primers 14a and 14b, template RFP in pZE21 miniprep
- 7,8,9 -> AraBAD promoter - Primers 12a and 12b, template biobrick K808000
Settings for PCR of 1,2,3
| Temperature (oC) | Time (min) | Rounds |
|---|---|---|
| 98 | 2:00 | 1 |
| 98 | 0:20 | 35 |
| 58 | 0:45 | 35 |
| 72 | 2:00 | 35 |
| 72 | 5:00 | 1 |
| 10 | ∞ | - |
Settings for PCR of 4-9
| Temperature (oC) | Time (min) | Rounds |
|---|---|---|
| 98 | 2:00 | 1 |
| 98 | 0:20 | 35 |
| 61 | 0:45 | 35 |
| 72 | 1:00 | 35 |
| 72 | 5:00 | 1 |
| 10 | ∞ | - |
Gel analyses of Nir operon, PCR product from 10-07-2013
We run 0,8% agarose gel with 9 samples.
PCR purification
PCR fragments for the Nir operon from todays PCR.
Result
0.8 % Agorase Gel to test yesterday's PCR of the Nir operon with different extension times
Wells
- 1: Nir extension time 2:00
- 2: Nir extension time 2:00
- 3: Nir extension time 2:00
- 4: Nir extension time 3:00
- 5: 1kb ladder
- 6: Nir extension time 3:00
- 7: Nir extension time 3:00
- 8: Nir extension time 4:00
- 9: Nir extension time 4:00
- 10: Nir extension time 4:00
Conclusion
We have now tried PCR of Nir more than one time and same result appear. This must then be the true chromosomal fragment but with some additional sequence in the middle. We will try to do another PCR with USER primers tomorrow to see if we then get same fragments or shorter ones.
Navigate to the Previous or the Next Entry
