Team:Brown/Notebook/October4

From 2010.igem.org

(Difference between revisions)
(New page: {{:Team:Brown/templates/header}} ==Monday, October 4, 2010== ===Digest and ligation of LacI into pSB1C3=== LacI (linear, blunt ends) will be double digested with PstI and EcoRI and insert...)
(→Monday, October 4, 2010)
Line 27: Line 27:
*10ul insert (LacI)
*10ul insert (LacI)
*2ul H2O
*2ul H2O
 +
 +
'''Digest AraC'''
 +
(2 Tubes)
 +
 +
50 μl total
 +
25 μl H2O
 +
5 μl 10x buffer H
 +
2.5 μl EcoRI
 +
25 μl Pst1
 +
15 μl DNA (AraC-RFC25)
 +
 +
37°C at 21:36
 +
out at 22:36
 +
PCR purification, eluted in 30μl
 +
 +
'''Ligation'''
 +
30 μl total
 +
15 μl buffer 2x
 +
2 μl ligase
 +
2 μl vector (psB1C3)
 +
11 μl insert (AraC)
 +
 +
in 4°C at 23:13

Revision as of 20:07, 24 October 2010

Monday, October 4, 2010

Digest and ligation of LacI into pSB1C3

LacI (linear, blunt ends) will be double digested with PstI and EcoRI and inserted into pSB1C3.

Digest

50ul total volume

  • 25ul dH2O
  • 5ul 10x buffer H
  • 2.5ul EcoRI
  • 2.5ul PstI
  • 15ul DNA

Ran 2 tubes in parallel. the dH2O used for the first tube had a plastic-y smell, so the tube was discarded and this was noted in case of later error

Cleanup

Followed Qiagen PCR cleanup protocol

Ligation

30ul total volume

  • 15ul 2x rapid buffer
  • 2ul ligase (T4)
  • 1ul vector
  • 10ul insert (LacI)
  • 2ul H2O

Digest AraC (2 Tubes)

50 μl total 25 μl H2O 5 μl 10x buffer H 2.5 μl EcoRI 25 μl Pst1 15 μl DNA (AraC-RFC25)

37°C at 21:36 out at 22:36 PCR purification, eluted in 30μl

Ligation 30 μl total 15 μl buffer 2x 2 μl ligase 2 μl vector (psB1C3) 11 μl insert (AraC)

in 4°C at 23:13

Retrieved from "http://2010.igem.org/Team:Brown/Notebook/October4"