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 resulting in 1107bp bands.
|
|
|
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 (VR iGEMGr09) out of 5 primers is not working.
- Isolation of pSB1AC3 + promoters and pSB3K3 + promoters to check constructs.
- Isolated the vectors from o/n culture
- Used colonies for o/n culture were:
- AC3-1: colony 3
- AC3-2: colony 10
- AC3-3: colony 11
- K3-1: colony 23
- K3-2: colony 26
- K3-3: colony 37
- Used Sigma plasmid isolation kit, eluted in 50ul MQ
DNA concentrations were:
| Sample
| ng/ul
| 260/280
| 260/230
|
| K3-1
| 6.8
|
|
|
--> TODO fill table
- PCR on plasmids to check insert size.
- Used pSB1K3 as negative control
- Used pipeting scheme
|
|
|
VR-VF2
| Component | amount
|
| MasterMix NH4
| 21 uL
|
| F primer
| 1 uL
|
| R primer
| 1 uL
|
| vector
| 1.0 uL
|
| Taq polymerase
| 1 uL
|
|
|
VR VF NIENKE program
| Stage
| Temperature
| Time
|
| Denaturing
| 95°
| 5 min
|
|
| Start Cycles 30X
|
| Denaturing
| 95°
| 20 sec
|
| Annealing
| 61°
| 20 sec
|
| Elongation
| 72°
| 20 sec
|
|
| End cycles
|
| Final elongation
| 72°
| 10 min
|
| Hold
| 4°
| Forever
|
|
- Keep in the fridge and check on Agarose gel tomorrow.
- Transform E. coli TOP10 with (pBAD promoter in pSB2K3) and (Lac promoter in pSB1A2)
- Use normal transformation protocol (17 June 2009)
- Also a negative control was taken, MQ.
- Plate the pBAD transformant on LB-Kan and the Lac transformant on TY-Amp.
- O/n @ 37dg
Dry
Jasper finished the GlpF model by fitting a Michaelis-Menten equation to the raw data (using the method of Goudar1999). Mathematica proved to be the best tool for this, as it is one of the few programs that has the Lambert W function built-in (as ProductLog). Furthermore, an interactive model was added to the Wiki demonstrating the results.
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