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Team:Brown/Notebook/September16
From 2010.igem.org
Thursday, September 16, 2010
Gel Purification
Followed 'QiaQuick PCR purification kit protocol (pg19 in manual). Eluted in 30ul for higher vol. Put purified digests back in the freezer in TatPTD box
Ligation of purified digest products (LacI, AraC) into opened, purified J13002 plasmid
Rxn A: 15ul total volume
- 7.5ul ligase buffer (rapid 2x)
- 1ul T4 DNA ligase
- 0.5ul vector (J13002)
- 3ul AraC (RFC25 purified digest insert)
- 3ul dH2O
Rxn B: 15ul total volume
- 7.5ul ligase buffer (rapid 2x)
- 1ul T4 DNA ligase
- 0.5ul vector (J13002)
- 3ul LacI (RFC25 purified digest insert)
- 3ul dH2O
In fridge(4C) overnight
Double digest of tat-linker brick, pBSsk with EcoRI and PstI
Result is to open up bluescript and prepare tat-linker for insertion. The appropriate TF will then be ligated in.
50ul total volume
- 5ul buffer H (Promega)
- 3ul DNA
- 1ul EcoRI
- 1ul PstI
- ul H2O
PCR purification/cleanup of EcoPst double digest
Re-eluted samples in 30ul of EB instead of 50 to up concentration
Nanodrop: TatL = 3.5ng/ul | pBS = 21.5ng/ul
Ligation of purified digest products (tat-linker) into open pBS plasmid
15ul total volume
- 7.5ul ligase buffer (rapid 2x)
- 1ul T4 ligase
- 0.5ul pBS vector DNA
- 6ul TatLinker insert DNA
in 4C overnight (start 4:30PM)
