Team:NTU-Singapore/Notebook/11 July 2008
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+ | =Friday 11 June= | ||
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*SupD gel check showed that PCR product was not SupD - re-ran the PCR and made sure the product was right this time. | *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. | *GFP-6 and E7 were digested. | ||
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*2010: QIA purify for pFe digested at 1910. After that, we ran gel to see and surprisingly, all the plasmids had clear and distinct 300bp bands without the signs of uncut plasmids (2.3kb). We then gel extracted for this pFe insert and stored at -20 under '''ready for ligation'''. | *2010: QIA purify for pFe digested at 1910. After that, we ran gel to see and surprisingly, all the plasmids had clear and distinct 300bp bands without the signs of uncut plasmids (2.3kb). We then gel extracted for this pFe insert and stored at -20 under '''ready for ligation'''. | ||
*2150: plate the LacI-GFP cells on agar plates. | *2150: plate the LacI-GFP cells on agar plates. | ||
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Latest revision as of 02:26, 28 October 2008
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Friday 11 June
Summary:
..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.
Lab details for ligation of LacI-GFP and Fe-GFP:
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