Team:Heidelberg/Notebook measure/NotebookDP

Dual plasmid

Contents

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