Team:Heidelberg/Notebook measure/NotebookDP

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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
--> ligation PCR_mCherry_CMV  and p31 can not have worked either
 * transfection of the ligation PCR_mCherry in p7 from 8-28-09 did not work, but positive control worked: ligation not successful
 * 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
--> Repeat from the beginning
 * 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


 * 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


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