Template:Team:KULeuven/1 September 2009/VanillinProduction
From 2009.igem.org
- SAMS I B RD is used for ligation with TER II RD
- Since we want to avoid gel purification, we use the direct sample from the restriction digest
- The restriction enzymes are denatured by keeping the sample at 85°C for 20 minutes
| vector | insert
|
| Terminator | SAMS
|
| 2000 bp | 3070 bp
|
| 50 ng | 280 ng
|
| 10 μl | 16 μl
|
- The genes fcs and ech are individually cut with the four restriction enzymes
| EcoR1 | fcs RD-E
|
| Spe1 | fcs RD-S
|
| Pst1 | fcs RD-P
|
| Xba1 | fcs RD-X
|
| EcoR1 | Ech RD-E
|
| Spe1 | Ech RD-S
|
| Pst1 | Ech RD-P
|
| Xba1 | Ech RD-X
|
- Results on gel show that all the genes have the same length and display 1 line.
- SAMS I A RD is put on gel, to be used for gel extraction and a part of SAMS I B RD is put on gel to check the length.
- Replated colonies from the original SAMS plate from 24/8
- Took 4 individual colonies and plated them on 4 corners of a petri dish
- the 4 genes(sam5, sam8, ech, fcs) where taken from the original DNA that was sent, electroporated and plated. This in order to make a glycerol stock.
- We also started another method to try and ligate the different genes for vanillin production by using PCR. Today we did PCR on the ech, fcs, sam5 and sam8 genes to get the DNA out of the plasmid. We used the prefix and suffix primers from last year, these primers keep the pre- and suffix of the biobrick part intact and allow us to work with the purified biobricks themself.
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