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Team:DTU-Denmark/Notebook/5 September 2013
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of HAO User (faint bands) and HAO ex (bright bands). HAO ex will be used as a template for new PCRs to amplify HAO User. | of HAO User (faint bands) and HAO ex (bright bands). HAO ex will be used as a template for new PCRs to amplify HAO User. | ||
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===PCR for HAO with USER endings=== | ===PCR for HAO with USER endings=== | ||
Revision as of 11:47, 6 September 2013
5 September 2013
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Contents |
Lab 208
Main purpose
PCR for HAO with USER endings after purification
Who was in the lab
Henrike, Ariadni
Procedure
Gel purification
of HAO User (faint bands) and HAO ex (bright bands). HAO ex will be used as a template for new PCRs to amplify HAO User.
PCR for HAO with USER endings
Template: HAO extraction fragment (purified from PCR run on 04.09)
Primers: 18a, 18b
Two versions, one with 5% DMSO and one with 3% DMSO and two programs: Touchdown (62C -> 55) and a single annealing temperature of 59C.
PCR reaction master mix (x5)
| compound | volume (uL) |
|---|---|
| dNTPs | 1 |
| HF | 10 |
| polymerase x7 | 0.5 |
| FW | 3 |
| RV | 3 |
| template | 1 |
| MQ | 29 |
| DMSO | 1.5 |
- Add 49 ul of the master mix, and
- Add 1 ul DMSO (for the 5%) or 1 ul of MQ (for the 3%)
Results
Gel on yesterdays PCR
- 2-log ladder
- HAO USER on 59C, 3% DMSO
- HAO USER on 59C, 3% DMSO
- HAO USER on 57C, 3% DMSO
- HAO USER on 57C, 3% DMSO
- HAO USER on touchdown
- HAO USER on touchdown
- HAO USER on touchdown, 3% DMSO
- HAO USER on touchdown, 3% DMSO
- HAO extraction fragment on 57C, template -20 culture
- HAO extraction fragment on 57C, template -20 culture
- HAO extraction fragment on 57C, template -80 culture
- HAO extraction fragment on 57C, template -80 culture
- HAO extraction fragment on 59C, template -20 culture
- HAO extraction fragment on 59C, template -20 culture
- HAO extraction fragment on 59C, template -80 culture
- HAO extraction fragment on 59C, template -80 culture
- 2-log ladder
Conclusion
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