Template:SJTU-BioX-Shanghai/Notebook/Construction/w3

SJTUB construction logo.jpg Construction

July 21

  1. Try different conditions of overlapping PCR for the whole construction of P_dark-HR* and pCPCG2-HR*.
  2. The final concentration of digestion product was too low for downstream operation. We suspected that it might be misoperation. Also we found out that we used isocaudarners XbaI and BcuI (also called SpeI) as digestion reaction sites, so we changed the two sites later.

July 22

  1. Try different conditions of overlapping PCR for the whole construction of P_dark-HR* and pCPCG2-HR*.
  2. Restriction enzyme digestion of pBluescript. CIAP was added in one sample and another one was not. Moreover, the reaction time was lengthened. We found out that CIAP didn’t seem to be that useful in our experiment. But longer reaction time was indeed needed.

July 23

  1. Try different conditions of overlapping PCR for the whole construction of P_dark-HR* and pCPCG2-HR*.
  2. We decided to make sure that which method of plasmid‘s digestion reaction is better, single digest or double digest and which enaturation way is better. So we designed several controls using the wrong enzymes as a precursive test. It’s also because that we need to purchase the new enzyme at the time.
    Digestion reaction pBluescript(D) pBluescript(S1) pBluescript(S2) P_dark-HR*/ pCPCG2-HR*
    ddwater 9μl 9μl 9μl Fill up to 30μl
    10×（green）buffer 2μl 2μl 2μl 2μl
    Plasmid/DNA fragment 7.2μl(1μg) 7.2μl(1μg) 7.2μl(1μg) 1μg
    BcuI 1μl 1μl 1μl 1μl
    XbaI 1μl 1μl 1μl 1μl
    Total volume 20μl 20μl 20μl 30μl
    Reaction 37℃ for 20mins 37℃ for 20mins 37℃ for 20mins
    Denaturation Gel extraction Purification Thermodynamic denauration

    Ligation P_dark-HR*(D)/ pCPCG2-HR*(D) P_dark-HR*(S1)/ pCPCG2-HR*(S1) P_dark-HR*(S2)/ pCPCG2-HR*(S2)
    ddwater Fill up to 10μl Fill up to 10μl Fill up to 10μl
    10×buffer 1μl 1μl 1μl
    Vector DNA 60ng 60ng 60ng
    Insert DNA 300ng 300ng 300ng
    Ligase 0.2μl(1WeissU) 0.2μl 0.2μl
    Total volume 10μl 10μl 10μl
    Reaction 17℃ for 4h 17℃ for 4h 17℃ for 4h
    Denaturation Purification/None Purification/None Purification/None
    Transformation.
    Results:
    pCPCG2-HR(D) pCPCG2-HR(S1) pCPCG2-HR(S2)
    Normal Normal Very few colonies
    P_dark-HR(D) P_dark-HR(S1) P_dark-HR(S2)
    Normal No colony Very few colonies