Team:Heidelberg/Notebook/BSDesign/October

From 2010.igem.org

(Difference between revisions)
(→10/05/2010)
Line 8: Line 8:
= Binding Site Design - October =
= Binding Site Design - October =
-
== 10/02/2010 ==
+
== 02/10/2010 ==
<br />
<br />
*digestion in 50 µl of  
*digestion in 50 µl of  
Line 14: Line 14:
**4x pSB1A3 (5µl BSA, 5µl NEB buffer 2, 1µl PstI, 1µl EcoRI, 1µl DpnI, 4µl construct, 33 µl H2O)
**4x pSB1A3 (5µl BSA, 5µl NEB buffer 2, 1µl PstI, 1µl EcoRI, 1µl DpnI, 4µl construct, 33 µl H2O)
-
== 10/05/2010 ==
+
== 05/10/2010 ==
<br />
<br />
*1µg digestion of miMeasure and 6 different constructs (1A, 3F, 13, 15, 17, 37) with EcoRI and PstI
*1µg digestion of miMeasure and 6 different constructs (1A, 3F, 13, 15, 17, 37) with EcoRI and PstI
Line 24: Line 24:
-
== 10/06/2010 ==
+
== 06/10/2010 ==
20 Miniprep:
20 Miniprep:
Line 80: Line 80:
-
== 10/12/2010 ==
+
== 12/10/2010 ==
4 miraPCRs for binding sites of miR4286.
4 miraPCRs for binding sites of miR4286.
Line 92: Line 92:
**transformation
**transformation
-
== 10/13/2010 ==
+
== 13/10/2010 ==
*pick colonies
*pick colonies

Revision as of 03:14, 28 October 2010
 


Binding Site Design - October

02/10/2010


  • digestion in 50 µl of
    • 4x pSB1C3 (5µl BSA, 5µl NEB buffer 2, 1µl PstI, 1µl EcoRI, 1µl DpnI, 4µl construct, 33 µl H2O)
    • 4x pSB1A3 (5µl BSA, 5µl NEB buffer 2, 1µl PstI, 1µl EcoRI, 1µl DpnI, 4µl construct, 33 µl H2O)

05/10/2010


  • 1µg digestion of miMeasure and 6 different constructs (1A, 3F, 13, 15, 17, 37) with EcoRI and PstI
1= 0S
2= 0L
3= 10S
4= 10L


06/10/2010

20 Miniprep:

Table 1: 20 Minis
Numberbinding site against miRNA xPosition
12213.5
22211.4
311793.1
442862.6
542864.1
642863.4
711794.4
83.5
911791.9
102214.6
112211.5
1242863.5
132214.8
142212.3
1542862.2
1642861.3
1711792.5
1811792.4
1911791.5
2011794.8
  • digestion of miMeasure and 6 different synthetic miRNA patterns against endogenous miR122
  • gel seperation
  • gel purification
  • sequencing


12/10/2010

4 miraPCRs for binding sites of miR4286.

  • one PCR with an annealing temperature of 45 degree and 16 cycles
  • second miraPCR with annealing temperature of 57 degree and also 16 cycles
    • nucleotide removal
    • digestion
    • gel seperation
    • gel purification
    • ligation in miMeasure and pSB1C3
    • transformation

13/10/2010

  • pick colonies
  • about 8 hours later: do minipreps
  • send for sequencing

Retrieved from "http://2010.igem.org/Team:Heidelberg/Notebook/BSDesign/October"