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Exeter/16 July 2013
From 2013.igem.org
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| + | ==So what went wrong with our digestion/ligation/tranformation?== | ||
| + | So our first try at attaching two BioBricks together didn't work as well as we hoped! But how do we know if something went wrong with our digestion, ligation or transformation? | ||
| + | |||
| + | We can assume that, as the RFP transformations worked, the problem is probably unrelated to the transformation stage. The digestion and/or ligation are more likely to be the culprits. Most protocols we have received from iGEM headquarters have been looked at by our supervisors, various academics and some of the PhD students we share the labs with, who "optimise" the protocols for the chemicals, enzymes and equipment we use in the lab. This can include changing the chemicals used in each stage, the timings of certain steps, or the equipment we use. However, we did not do this with the digestion and ligation protocols, we simply followed the ones from iGEM HQ. | ||
| + | |||
| + | Obviously we didn't have the products from our digestion to hand; they had been used in the ligation steps. But we did have some of the products from our ligation stage, so we decided to run these on a gel to see where we went wrong. | ||
| + | |||
| + | The gel used is a standard TBE gel, made using the instructions found on 8/7/13. The mastermix for the gel contained... | ||
| + | |||
| + | *120ul distilled water | ||
| + | *20ul buffer (includes a dye) | ||
| + | *5ul XbaI | ||
| + | *5ul SpeI | ||
| + | |||
| + | The XbaI and SpeI should cut our New Parts from their plasmids, so when we run the gel, we should see two bands per lane; one for the plasmid and one for the New Part. | ||
| + | |||
| + | '''Ligation Gel''' | ||
Revision as of 14:43, 18 July 2013
So what went wrong with our digestion/ligation/tranformation?
So our first try at attaching two BioBricks together didn't work as well as we hoped! But how do we know if something went wrong with our digestion, ligation or transformation?
We can assume that, as the RFP transformations worked, the problem is probably unrelated to the transformation stage. The digestion and/or ligation are more likely to be the culprits. Most protocols we have received from iGEM headquarters have been looked at by our supervisors, various academics and some of the PhD students we share the labs with, who "optimise" the protocols for the chemicals, enzymes and equipment we use in the lab. This can include changing the chemicals used in each stage, the timings of certain steps, or the equipment we use. However, we did not do this with the digestion and ligation protocols, we simply followed the ones from iGEM HQ.
Obviously we didn't have the products from our digestion to hand; they had been used in the ligation steps. But we did have some of the products from our ligation stage, so we decided to run these on a gel to see where we went wrong.
The gel used is a standard TBE gel, made using the instructions found on 8/7/13. The mastermix for the gel contained...
- 120ul distilled water
- 20ul buffer (includes a dye)
- 5ul XbaI
- 5ul SpeI
The XbaI and SpeI should cut our New Parts from their plasmids, so when we run the gel, we should see two bands per lane; one for the plasmid and one for the New Part.
Ligation Gel
