Team:Heidelberg/Notebook/miRNA Kit/September

02/09/2010

• cloning of different shRNAs into pcDNA5/FRT/TO
• 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