quick and dirty cloning of tuning and tetR construct goes on
03/09/2010
... and on
04/09/2010
mini-prep of pcDNA5 with shRNAs 1, 2, 3, 5, 5* and 6 (each four colonies from two independent transformations)
PCR of shRNA constructs (including shRNA 7, 8 and 9 as well)
gel: fine, everywhere amplified sequences below 200 bp
digestion of promising tuning constructs with BspEI and StuI
gel: plasmids seem to be cut only once again (problem maybe BspEI), size is correct
suggestion: another digestion on monday with SalI and KpnI, then: submission for sequencing
06/09/2010
digestion of tuning construct with KpnI and SalI result in weird band pattern on the gel
PCR and purification of the binding sites looked fine on the gel
submission of tuning constructs and pcDNA5 containing different miRNAs for sequencing
07/09/2010
repetition of TetR and tuning construct cloning
purification and digestion of PCR amplified binding sites with NotI
08/09/2010
cloning of shRNAs in pcDNA5 again
further digestion of binding sites with AscSI (analog to SgfI) and purification
preparation of psyCHECK2 for insertion of binding sites and shRNAs for first test measurements
transformation of TetR constructs
ligation of both inserts for the tuning construct, extraction of the right insert (3000bp) and ligation into the backbone
09/09/2010
colony PCR of TetR constructs are positive
inoculate TetR for miniprep
10/09/2010
miniprep of TetR
test digest of TetR with BspEI & NotI and with SgfI gave promising results
YES it was done with marker instead of loading dye but:
for lanes 2-4 you see a stronger band at 700 pb (size of TetR)
for landes 5-7 you see a stronger band at 5000 bp as it was linearized with SgfI
colony PCR of binding site in PsiCheck and tuning construct.. tuning construct looks promising, BS nothing on the gel except for primers
colony PCR of shRNAs look promising even though we cannot say whether still the old shRNA is inside
PCR of CMV shRNA which leads to a size of 1200bp
11/09/2010
miniprep of tuning construct and assorted shRNA BS PsiCheck colonies. Repetition of test PCR (for tuning primer L11 and L13, for BS primer L1 and 86a (dominik)) with miniprep template. Tuning construct looks kind of good on the gel, but I don't want to get you hopes up too soon.. it will be send for sequencing on monday.
Tet Repressor cloned!!!
shRNA 2,3,5,7,8 and 9 seems to be cloned according to test digest
12/09/2010
miniprep of shRNA constructs and Tuning constructs
13/09/2010
digestion and cloning of binding sites and PsiCheck.. again
submission of constructs for sequencing
14/09/2010
note: for sequential digest of pcDNA5 or shRNAs with ApaI and HindIII use buffer 2
15/09/2010
digestion of TetR construct with AsiSi and NotI or SgfI and NotI respectively
for cloning of binding sites for miR122, miR375 and miR376
ligation of the other binding sites for synthetic pri-miRNA (6, 7, 8, 9 and 10) with digested psiCHECK2
16/09/2010
PCR to generate binding site oligos, nucleotid removal, digestion, nucleotid removal
different digestion and ligation protocols tested for binding sites and psiCHECK2, afterwards transformation
possibilities:
dilution of oligo insert [1:100,1:500], ratio: insert/vector = 5/1
SAP treatment for oligos to prevent them from forming concatemers
SAP treatment of backbone to prevent it from re-ligation
cut of backbone into two fragments to ease gel excision (if done) and advance ligation
ligation process: 1h @ RT or over-night @ 4°C in darkness
note: bs122 confused with bs7
17/09/2010
colony PCR, mini-prep and digestion of promising psiCHECK or TetR constructs containing binding site
control plates with horrible number of re-ligations
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