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Team:Heidelberg/Tyrocidine week12 ms
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===Validation of the strain from Marahiel by Colony PCR=== | ===Validation of the strain from Marahiel by Colony PCR=== | ||
====Primers used==== | ====Primers used==== | ||
Revision as of 22:33, 2 October 2013
Contents |
Validation of the strain from Marahiel by Colony PCR
Primers used
TycA_A-domain: IK09, IK10
TycB1_A-domain: IK11, PW01
TycB1_E-domain: PW02, PW03
Conditions
File:B.brevis-colony-PCR2013-07-18.png
Colony-PCR of B. brevis. Lane 4: NEB 2-log; lane 1: primers IK09+IK10; lane 2: primers IK11+PW01;lane 3: primers PW02+PW03
| Cycles | temperature [°C] | Time [s] |
|---|---|---|
| 1 | 95 | 300 |
| 12 | 95 | 60 |
| 66 ↓0.5°C | 30 | |
| 72 | 240 | |
| 23 | 95 | 60 |
| 60 | 30 | |
| 72 | 240 | |
| 1 | 72 | 600 |
| 1 | 12 | inf |
Handling of the strain
- received Brevibacillus parabrevis ATCC 8185 from Marahiel lab
- plate stood 1 week at RT in Marburg => wet, stinks
- plate cells on LB, inoculate 2x YT medium
- scratch everything from plate, centrifuge, wash with H2O, centrifuge, resuspend in H2O
Result
Three bands were visible in the UV-chamber. The three respective lanes implicate a fragment size of 1.5kb, 1.5kb and 1.2kb. This matches well the expected sizes. Therefore we can conclude that our colony PCRs have been successful!
Date: 19-07-2013
Evaluation of the strain
Result
Colonies grew over night -> as colony PCRs were positive and the strain was cultivatable, the plate of marburg seemed to be intact (despite long storage under bad conditions).
