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Team:NTU-Singapore/Notebook/11 July 2008
From 2008.igem.org
(New page: *SupD gel check showed that PCR product was not SupD - re-ran the PCR and made sure the product was right this time. *GFP-6 and E7 were digested. *Gel checks were also ran for T7ptag & E...) |
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*Purification problems.. | *Purification problems.. | ||
| - | ..were solved with the addition of 10μl of Proteinase K to 50μl of PCR product, then incubated at 37degC for 30mins, then 68degC for a further 10 minutes. This yielded clear bands that were otherwise smeary and thus indistinguishable. | + | ..were solved with the addition of 10μl of Proteinase K to 50μl of PCR product, then incubated at 37degC for 30mins, then 68degC for a further 10 minutes. This yielded clear bands that were otherwise smeary and thus indistinguishable. Addition of proteinase K also increase the yield of PCR purification, DNA concentration jumped from previous values of 40s to 300s. Most probable reason for such phenomenon is that Proteinase K being a protein degrading enzyme is able to degrade any residual TAQ polymerase that are still bounded to the DNA that is causing inefficient binding of DNA to the purification membrane. |
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| + | Novel Idea proposed by Prof Matthew: | ||
| + | Due to the problem of uncut vectors, prof chang proposed to use miniprep membrane which is able to hold circular membrane only to purify gel extracted products so that elution volume would contain linearised plasmids and with a furthur step of PCR purification of elution volume, we will be able to obtain only linearised plasmids, therefore increasing the chances and efficiency of ligation. Theory was proven wrong. Gel checks of before and after elution volume showed negative results. | ||
*Gel extraction of E7 & T7ptag (E-P) was also carried out. | *Gel extraction of E7 & T7ptag (E-P) was also carried out. | ||
Revision as of 03:56, 15 July 2008
- SupD gel check showed that PCR product was not SupD - re-ran the PCR and made sure the product was right this time.
- GFP-6 and E7 were digested.
- Gel checks were also ran for T7ptag & E7. Darius found out that running SupD on a 1.5% agarose gel, compared to the usual 1% gel would yield clearer bands - this proved to be true.
- Purification problems..
..were solved with the addition of 10μl of Proteinase K to 50μl of PCR product, then incubated at 37degC for 30mins, then 68degC for a further 10 minutes. This yielded clear bands that were otherwise smeary and thus indistinguishable. Addition of proteinase K also increase the yield of PCR purification, DNA concentration jumped from previous values of 40s to 300s. Most probable reason for such phenomenon is that Proteinase K being a protein degrading enzyme is able to degrade any residual TAQ polymerase that are still bounded to the DNA that is causing inefficient binding of DNA to the purification membrane.
Novel Idea proposed by Prof Matthew: Due to the problem of uncut vectors, prof chang proposed to use miniprep membrane which is able to hold circular membrane only to purify gel extracted products so that elution volume would contain linearised plasmids and with a furthur step of PCR purification of elution volume, we will be able to obtain only linearised plasmids, therefore increasing the chances and efficiency of ligation. Theory was proven wrong. Gel checks of before and after elution volume showed negative results.
- Gel extraction of E7 & T7ptag (E-P) was also carried out.
- GFP (X-P) & E7 (X-P) produced no bands.
- Gel run for LacI-GFP & Fe-GFP
- Gel check for re-digested Fe.
- Ligation
- Two protocols were carried out, one was Drew Endy's protocol (using T4 DNA Ligase) and the other was Quick Ligase, both on E7 and T7ptag.
