Team:Heidelberg/Notebook measure/NotebookDP

From 2009.igem.org

Dual plasmid

Contents

Week Days
35 - - - 8-27-2009 8-28-2009 - -
36 - 9-01-2009 9-02-2009 9-03-2009 9-04-2009 9-05-2009 9-06-2009
37 9-07-2009 - - 9-10-2009 - - -


8-27-09

  • restriction digest of p7 w/ NheI and BclI
  • restriction digest of PCR_mCherry (from p43 with O.50 and O.171) with AvrII and BclI
  • preperative gel electrophoresis: digest with BclI didn't work--> redo
    • restriction digest of p7 w/ NheI and PCR-mCherryy w/ AvrII; heat-inactivated both enzymes
    • BclI can not be heat-inactivated!

8-28-09

  • restriction digest of p7 and PCR_mCherry w/ BclI for 2 hours at 50°C
    • looked at result on agarose gel: 3 clear bands, where only two are suppossed to be
  • 2 Strategies:
    • cut out all 3 bands, gel purify them and ligate them with digestet PCR_mCherry
    • redo BclI digest later

9-01-09

  • Transformation of PCR_mCherry_p7
    • should have been in unmethylating cells, but wasn't--> no test-digest with BclI possible
    • control result via PCR--> Product can be used for further cloning

9-02-09

  • picked colonies from transformation of PCR_mCherry_p7
  • PCR of minipreps from ligation PCR_mCherry_p7 with O.172 and O.173 to amplify CMV_mCherry with SphI restriction sites on both ends

9-03-09

  • redid Bcl1/Nhe1 digest of p7: at least 9 clear bands visible on 1% argarose gel--> has to be redone again
  • PCR purification of PCR_mCherry_CMV ( 8 samples from ligation)
  • restriction digest of PCR_mCherry_CMV ( 8 samples from ligation) with SphI
  • restriction digest of p31 with Sph1
  • gel purification of PCR_mCherry_CMV ( 8 samples from ligation) and p31
    • 4 clear bands in all samples where only 2 are suppossed to be, but one band w/ the right length; reason: probably because melting temperatures of the primers differed too much
      • gradient pcr to check if only one band would appear at the right conditions - without success. No further bands were cut out
    • cut out band with the right length from to lanes and one longer band from one lane
  • ligation of PCR_mCherry_CMV and p31
  • transformation of the ligation PCR_mCherry_CMV and p31
  • transfection of the ligation PCR_mCherry in p7 from 8-28-09 in HeLa cells according to effectene protocol

9-04-2009

  • transfection of the ligation PCR_mCherry in p7 from 8-28-09 did not work, but positive control worked: ligation not successful

--> ligation PCR_mCherry_CMV and p31 can not have worked either

  • new start from the beginning:
    • restriction digest of p7 with Bcl1 at 50°C for 1 hour
    • PCR-purifiction of the digest and redilution in 30 µl water
    • restriction digest of p7_Bcl1 with nhe1 at 37°C for 1 hour
  • preparative gelelectrophoresis
  • extraction of Jet with MfeI and NheI restriction sites from p31 by PCR (O.203 and O.71)
  • Maxiprep of p53
    • sent for sequencing

9-05-2009

  • gel purification of MfeI_Jet_NheI
  • restriction digest of MfeI_Jet__NheI and p6 with MfeI and NheI
  • preparative gelelectrophoresis
    • the lane of MfeI_Jet__NheI contained no DNA

--> Repeat from the beginning

  • picked 8 colonies of the latest p7::mcherry transformation

9-06-2009

  • Miniprep of p7::mcherry
    • extraction of CMV::mcherry from p7::mcherry by PCR with O.172 and O.173
  • extraction of Jet with MfeI and NheI restriction sites from p31 by PCR (O.203 and O.71)

9-07-2009

  • PCR-purification of PCR_mCherry_CMV and PCR_Mfe_Jet_Nhe
  • restriction digest of p31 and PCR_mCherry_CMV with Sph1
  • restriction digest of p7 and PCR_Mfe_Jet_Nhe with MFe1 and Nhe1
  • preparative gelelectrophoresis of the restriction digests

9-10-2009

  • ligated mCherry (digested w/ BclI, AvrII) with p7 (BclI, NheI) and p7JeT (BclI, NheI) overnight