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Team:University of Ottawa/21 July 2008
From 2008.igem.org
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::<li>I then proceeded to digest each sample with NcoI and run them on a gel for confirmation. | ::<li>I then proceeded to digest each sample with NcoI and run them on a gel for confirmation. | ||
::<li>It appeared as though NcoI only cleaved at one site on the plasmid - linearizing the plasmid. I am going to try to attempt another digestion with EcoRV/EagI which should give two definitive bands - if successful then I will PCR amplify with SLN1 primers for integration into yeast. | ::<li>It appeared as though NcoI only cleaved at one site on the plasmid - linearizing the plasmid. I am going to try to attempt another digestion with EcoRV/EagI which should give two definitive bands - if successful then I will PCR amplify with SLN1 primers for integration into yeast. | ||
| + | '''Chris''' | ||
| + | :'''Transformation of Competent Cells''' | ||
| + | ::<li>Using ligated AtCRE that Dan prepared 22 July 2008, performed a transformation of competent cells according to protocol | ||
| + | ::<li>Performed transformation on a single sample only, as success was not expected. Used LB plates with ampicillin. Incubated against a control plate with no e. coli. | ||
| + | ::<li>Incubated overnight. | ||
Revision as of 15:19, 28 July 2008
Contents |
Today in the lab
Dan
- Gel of sample 2T
- For some odd reason all of the DNA stayed in the well... Could be some sort of salt contamination from NEB buffer 1, or a very large autoligation product. I will test the former by doing PCR cleanup and switching to ligation buffer and then run the gel again.
- Digestion of 1A 0B and both S and D
- The above DNA fragments were digested with PstI and SphI in Neb buffer 1, enzymes were denatured after an hour
- PCR cleanup was performed on the digested products, eluted with 50 uL water.
- Ligation
- Ligation of the DNA obtained from PCR cleanup was run overnight at 16 C
Matt
- Miniprep
- I performed a miniprep of the inoculated PTP2 plasmids and yielded good concentrations for all samples.
- I then proceeded to digest each sample with NcoI and run them on a gel for confirmation.
- It appeared as though NcoI only cleaved at one site on the plasmid - linearizing the plasmid. I am going to try to attempt another digestion with EcoRV/EagI which should give two definitive bands - if successful then I will PCR amplify with SLN1 primers for integration into yeast.
Chris
- Transformation of Competent Cells
- Using ligated AtCRE that Dan prepared 22 July 2008, performed a transformation of competent cells according to protocol
- Performed transformation on a single sample only, as success was not expected. Used LB plates with ampicillin. Incubated against a control plate with no e. coli.
- Incubated overnight.
