From 2009.igem.org
Wet
GVP Cluster
Transporters
HmtA
The band at ~1150 kb was cut out of the gel and used for pcr described yesterday gave poor results. No band of expected size and strong primerdimer bands
Therefor we will redesign the cloning. The Forward primer will be modified into 2 part. which will require an additional PCR but increases the chance of getting product.
Metal Accumulation
Vectors
Positive control for the vector with insert done with both new and last years VR VF2 primers.
|
|
|
VR-VF2
| Component | amount
|
| MasterMix NH4
| 21 uL
|
| F primer
| 1 uL
|
| R primer
| 1 uL
|
| P3 vector
| 0.5 uL
|
| Taq polymerase
| 1 uL
|
|
|
Primers
| Cup nr
| F primer
| R primer
|
| 1
| VF2 iGEMGr09
| VR iGEMGr09
|
| 2
| VF2 iGEMGr09
| VR iGEMGr08 Aug
|
| 3
| VF2 iGEMGr09
| VR iGEMGr June
|
| 4
| VF2 iGEMGr08
| VR iGEMGr09
|
| 5
| VF2 iGEMGr08
| VR iGEMGr08 Aug
|
| 6
| VF2 iGEMGr08
| VR iGEMGr June
|
|
|
VR VF2 program
| Stage
| Temperature
| Time
|
| Denaturing
| 95°
| 5 min
|
|
| Start Cycles 30X
|
| Denaturing
| 95°
| 20 sec
|
| Annealing
| 60°
| 20 sec
|
| Elongation
| 72°
| 1.20 min
|
|
| End cycles
|
| Final elongation
| 72°
| 10 min
|
| Hold
| 4°
| Forever
|
|
This image shows 4 positive results, just the same as yesterdays but slighty higher bands. The above noted PCR was made again only this time without DNA, gel shown below. This indicates that one out of 5 primers is not working.
Dry
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