Team:NTU-Singapore/Notebook/11 July 2008



  

=Friday 11 June= {|border="1" style="background-color:#ffffcc;" cellpadding="20"

Summary:

 * 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.

..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.
 * Purification problems..

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.

=Lab details for ligation of LacI-GFP and Fe-GFP:=
 * 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.
 * We used front insert ligation: pLacI and pFe were inserts; GFP was vector. Therefore, we need to digest GFP with XbaI and EcoRI (sequential digestion); pLacI and pFe were digested with EcoRI and SpeI (double digestion).
 * 830-900: digest 1 ug GFP plasmid with XbaI.
 * 900: run a test digestion of pLacI with EcoRI and SpeI in Buffer 2 (instead of EcoRI buffer), but incubate for 1 hour only. The rationale behind this is due to the fact that both EcoRI and SpeI have 100% efficiency in Buffer 2, while SpeI is only 25% efficient in EcoRI buffer. However, EcoRI buffer is recommended by NEB for all double digestion including EcoRI because of star activity of this enzyme in other buffers. That's why we only let the digestion last for 1 hour only.
 * 1000: MinElute PCR purification for the above test digestion mixture. 10ul of digested pLacI obatined was then stored at -20 degrees.
 * 1200: MinElute for 1st digestion mixture of GFP.
 * 1245: 2nd digestion of 10ul after MinElute with EcoRI (in EcoRI buffer). Incubate for 3 hours (until 1600)
 * 1345: double digestion for pLacI and pFe (both with EcoRI and SpeI). Incubate for 2 hours (until 1600).
 * 1600: MinElute PCR purification for digested GFP,pLacI and pFe.
 * 1645: gel electrophoresis for the above digested plasmids. Results:
 * GFP vectors have clear 2.8 kb band. This band is alter gel extracted.
 * pLacI insert has a band at 250 (not too bright) which would be extracted later, a 2kb band, as well as a bright band at 2.3kb (uncut plasmid).
 * pFe insert only has a 2.3 kb band. No band at 300bp (pFe insert) was observed.
 * The pLacI digested with E/S in Buffer 2 for 1 hour, surprisingly, has very clear and distinct 250bp and 2kb bands. In addition, no sign of uncut plasmid (2.3kb) was observed. This result shows that the plasmids were not digested effectively with EcoRI and SpeI in EcoRI buffer (although incubated for 2hrs). Instead, by using Buffer 2, we could digest almost completely and also shorten the digestion time.
 * 1820: gel extraction
 * 1910-2010: cut pFe with E/S in Buffer 2 and incubate for 1 hour. If later gel check shows clear band at 300bp, we can extract it as pFe insert.
 * 1915: ligation of LacI with GFP using 1:3 ratio. Remaining amounts of digested LacI and GFP were stored in -20 degrees under Ready for ligation
 * 1935: MinElute for ligation mixture.
 * 2000: transform into top10 cells using 1,3 and 4 ul respectively.
 * 2030: heat shock and adding of SOC
 * 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.