Team:Heidelberg/Notebook/BSDesign/October

From 2010.igem.org

(Difference between revisions)
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*send for sequencing
*send for sequencing
{{:Team:Heidelberg/Single_Bottom}}
{{:Team:Heidelberg/Single_Bottom}}
 +
 +
----
 +
 +
= 14/10/2010 =
 +
* a reasonable
 +
* Screening of 40 colonies of miR4286 (colony PCR)
 +
* Screening of 32 colonies of miR320b (colony PCR)
 +
 +
 +
all negative
 +
<br /><br /><br />
 +
 +
----
 +
 +
= 15/10/2010 =
 +
 +
ligation of miMeasure construct:<br /><br />
 +
 +
miR-122<br />
 +
miR-122ran<br />
 +
miR-1179<br />
 +
miR-221<br />
 +
<br />
 +
each in 0S/0L/10S/10L versions
 +
 +
<br /><br />
 +
 +
----
 +
 +
= 16/10/2010 =
 +
transformation of each ligation to TOP10 ''E.coli''
 +
 +
cells were plated on LB-amp plates
 +
<br /><br /><br />
 +
 +
----
 +
 +
= 17/10/2010 =
 +
 +
96 colony PCRs - 8 from each plate
 +
<br>
 +
ALL NEGATIVE!!!!
 +
<br /><br /><br />
 +
 +
----

Revision as of 03:54, 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

14/10/2010

  • a reasonable
  • Screening of 40 colonies of miR4286 (colony PCR)
  • Screening of 32 colonies of miR320b (colony PCR)


all negative


15/10/2010

ligation of miMeasure construct:

miR-122
miR-122ran
miR-1179
miR-221

each in 0S/0L/10S/10L versions



16/10/2010

transformation of each ligation to TOP10 E.coli

cells were plated on LB-amp plates


17/10/2010

96 colony PCRs - 8 from each plate
ALL NEGATIVE!!!!


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