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
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
|
|
"
