E7 Story continued...

• Ethidium Bromide Gel showed clean bands at 2k mark, took PCR direct to PCR purified to 30ul.
  • Gel run-smeary band...What went wrong?

• Trouble SHOOT SHOOT
  • Protocol different from 8 July
    • Optimize Protocol

HOW? Identified potential causes of this problem as:

• Sequence of protocols ran - should we run a gel extraction before PCR purification? Or vice-versa?
• Loading amount during gel electrophoresis
• Ratio of loading dye to sample

THUS.. We ran a gel with the following lane contents for comparison.

AND WE FOUND ...that running a PCR purification AFTER gel extraction yielded the nicest (i.e. clearest, least smeary) band - yay! Best loading amount is 8microlitres and the loading dye issue is a non-issue.

T7ptag Story 2

PCR product obtained..Ran Gel with 4ul..Good clear band Ran gel to purify the rest..loaded with 10ul

• Hung:
  • 1030: gel run for GFP (digested with X/P on Tuesday). (done by Darius)
  • 1034-1100: QIA PCR purification for E7 (E/P) to concentrate E7 from 100ul into 30ul. After, nanodrop showed a concentration of 30ng/ul and A260/280 of 1.97.
  • 1125-1140: digestion of E7 with EcoRI and PstI to create sticky ends with following amounts (in ul):

E7: 26.4
EcoRI: 2
PstI: 2
EcoRI Buffer:4
BSA: 0.4
H2O: 5.2

Incubate for 3 hours (until 1500). At 1510, I also did PCR purification for E7, and used nanodrop to measure the concentration to calculate for ligation mixture. Then I carried out quick ligation for E7 with empty plasmid (however, nanodrops showed strange negative results of the ligation mixture). Transformation into the cell was carried out by Lu Chao from 1630-1800.

• • 1220-1320: Darius: gel extraction for supD and T7ptag. Nanodrops later showed insignificant concentration of supD after gel extracted. The T7ptag was kept for later ligation with empty vector from GFP.
  • 1350-1410: Min: PCR purification of supD, T7ptag (after gel extraction as above) and also PCR supD and T7ptag (w/o gel extraction). Nanodrops showed that both concentrations and purities of the PCR samples w/o gel extraction are good. We expected that considerable amounts of oligos were washed away by this purification kit. Darius would later run a gel to check whether the oligos were effectively removed from the PCR samples or not. Nanodrops for supD (with gel extraction before) showed weird datas, so we decided not to use that for ligation.
  • 1500-1820: digestion of T7ptag (with gel extraction), PCR supD and T7ptag (w/o gel extraction) with EcoRI and PstI for ligation with empty plasmids. Incubate for 3 hours.
  • 1830-1930: the following tasks were performed for the ligation of the above 3 digested plasmids with empty plasmid from GFP:
    • PCR purification to remove the enzymes, buffers...
    • Nanodrop to check concentrations. (quite desirable results).
    • Ligation
    • PCR purification again to concentrate the ligation mixtures. Then, nanodrops for checking. (done by Lu Chao).
  • 1945-2130: transformation into homemade top10 (done by Lu Chao).
• Lu Chao:
  • 1200-1630: Lu Chao prepared new set of LBA (including 4 hours autoclaving at 121 degrees).
  • 1700-1800: inoculation for LacI-RBS, LacI-GFP, Fe-GFP.