Hung, Darius, Choon Kit
try out ligation of pFe(insert) with GFP(vector); and pLacI(insert) with RBS34(vector). 300 ng is used for each DNA (the volume is calculated based on concentration measured by Nanodrop on Monday).
- 1030-1130:prepare digestion mixtures for pFe,GFP,pLacI,RBS34. Use double digestion for the inserts and sequential digestion for the vectors.
- Vectors (1st digestion):incubate from 1130-230pm at 37 degrees C
|
| GFP
| RBS34
|
| DNA
| 12.6
| 13.7
|
| Buffer2
| 2
| 2
|
| BSA
| 0.2
| 0.2
|
| Mq water
| 4.2
| 2.2
|
| XbaI
| 2
| 2
|
| Total
| 21
| 20
|
- Inserts (double digestion):incubate from 1130-130pm at 37 degrees C
|
| pLacI
| pFe
|
| DNA
| 27
| 16.5
|
| EcoRI Buffer
| 2
| 2
|
| BSA
| 0.2
| 0.2
|
| Mq water
| 0
| 10
|
| EcoRI
| 1
| 1
|
| SpeI
| 1
| 1
|
| Total
| 31
| 30
|
- 130pm-2pm: PCR purification (using PCR purification MinElute Kit) for pLacI,pFe to remove the enzymes (prevent star activity). After that, store the samples at 4 degrees fridge.
- 2pm-230pm: PCR purification (using PCR purification MinElute Kit) for GFP,RBS34 to remove the enzymes (prevent star activity)
- 230pm-6pm: 2nd digestion for GFP,RBS34 (including incubation for 3 hours). After this, we heated the DNAs at 80 degrees C for 20 mins to stop the enzymes reaction,then stored at 4 degrees fridge.
|
| GFP
| RBS34
|
| DNA
| 10 (after Minelute)
| 10 (after Minelute)
|
| EcoRI Buffer
| 2
| 2
|
| Mq water
| 6
| 6
|
| EcoRI
| 2
| 2
|
| Total
| 20
| 20
|
- Run the gel electrophoresis for newly synthesized pFe,pLacI,GFP,RBS32,RBS34,pT7,Terminator (show expected size) and pFe-GFP (show wrong 500 bp bands).
Choon Kit, Luchao
Chin Chong
- Cells that were left to grown overnight from 30/06/08 were removed from the incubator
- The cells were diluted with LB medium
- OD measurement was taken after dilution
- Cells were allow to an obsorbance of OD 1.2
- After which the cells were centrifuged and resuspended with M9 medium containing glycerol
- The cells in M9 medium were allowed to incubate at 37 deg for about 10 mins
- Once the cells were prepared, the characterization experiment were carried out as per before
- This time round however, we tested with the Low, Medium and High Promoters with GFP device that were sent to us in the first edition of the iGEM Newsletter, the standard promoter was also measured
- The plate reader was allowed to run for 12 hrs collecting RFU readings to be used to co-relate with each other
- Meet up with the Sales Engineer from ITS Science and Medical, to ask more about the plate reader that we are using for characterization. Questions shall be forwarded to the Product Engineer
- To send out Business Proposal to ITS Science and Medical
|
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