Team:HokkaidoU Japan/Notebook/August24

From 2010.igem.org

(Difference between revisions)
(New page: {{Template:HokkaidoU_Japan}})
 
(11 intermediate revisions not shown)
Line 1: Line 1:
-
{{Template:HokkaidoU_Japan}}
+
{{Template:HokkaidoU_Japan}}<div class="linkbar"><div class="prev">[[Team:HokkaidoU_Japan/Notebook/August23|August 23]]</div>[[Team:HokkaidoU_Japan/Notebook|Notebook]]<div class="next">[[Team:HokkaidoU_Japan/Notebook/August25|August 25]]</div></div>
 +
 
 +
=Check to see if digestion visualization Primers Work=
 +
 
 +
[[Image:HokkaidoU Japan 20100824a.jpg|200px|right|thumb|Electrophoresis after restriction enzyme digestion‎]]
 +
 
 +
* All 4 of PCR products were purified via Microcon YM-10
 +
 
 +
{| class="protocol"
 +
|'''Lane'''
 +
|'''DNA'''
 +
|-
 +
|1
 +
|TSUDA Marker I
 +
|-
 +
|3
 +
|RBS
 +
|-
 +
|4
 +
|GFP
 +
|-
 +
|5
 +
|double terminator
 +
|-
 +
|6
 +
|Promoter
 +
|}
 +
Promoter band in lane 6 wasn't visible
 +
* This vector didn't have a primer annealing site for our new primers
 +
 
 +
=Check of Ligation Mixtures=
 +
TAKARABIO's Ligation Mixture [I] and Ligation Mixture Mighty Mix [M]
 +
* After PCR and gel extracted pSB1C3 volume of either Ligation Mixture equal to pSB1C3 solution volume was added
 +
* Transformed
 +
* incubated in 200 uL of LB medium
 +
* 100 uL of each solution was plated on mediums containing 34 ug/uL and 12 ug/uL of chloramphenicol
 +
 
 +
=Various Checks=
 +
 
 +
[[Image:HokkaidoU Japan 20100824b.jpg‎|200px|right|thumb|Electrophoresis to check if various DNA manipulations were successful]]
 +
 
 +
{| class="protocol"
 +
|'''Lane'''
 +
|'''DNA'''
 +
|-
 +
|2
 +
|[https://2010.igem.org/Image:HokkaidoU_Pictures_DNA_Marker.png λ/''Hin''d III]
 +
|-
 +
|3
 +
|Ligation between pSB1C3 vectors
 +
|-
 +
|4
 +
|Product obtained by using digestion visualization primer for 1-2N
 +
|-
 +
|5
 +
|Flow through of digestion visualization primer for 1-2N
 +
|-
 +
|6
 +
|[https://2010.igem.org/Image:HokkaidoU_Pictures_DNA_Marker.png λ/''Hin''d III]
 +
|-
 +
|7
 +
|Gel extracted pSB1C3
 +
|}
 +
 
 +
Band weren't visible in lane 3
 +
* Dimers, trimers and etc were hardly visible maybe severely diluted
 +
 
 +
In lane 7 had detectable by very weak band
 +
* There weren't any problems with gel extraction

Latest revision as of 07:44, 27 October 2010

Check to see if digestion visualization Primers Work

Electrophoresis after restriction enzyme digestion‎
  • All 4 of PCR products were purified via Microcon YM-10
Lane DNA
1 TSUDA Marker I
3 RBS
4 GFP
5 double terminator
6 Promoter

Promoter band in lane 6 wasn't visible

  • This vector didn't have a primer annealing site for our new primers

Check of Ligation Mixtures

TAKARABIO's Ligation Mixture [I] and Ligation Mixture Mighty Mix [M]

  • After PCR and gel extracted pSB1C3 volume of either Ligation Mixture equal to pSB1C3 solution volume was added
  • Transformed
  • incubated in 200 uL of LB medium
  • 100 uL of each solution was plated on mediums containing 34 ug/uL and 12 ug/uL of chloramphenicol

Various Checks

Electrophoresis to check if various DNA manipulations were successful
Lane DNA
2 λ/Hind III
3 Ligation between pSB1C3 vectors
4 Product obtained by using digestion visualization primer for 1-2N
5 Flow through of digestion visualization primer for 1-2N
6 λ/Hind III
7 Gel extracted pSB1C3

Band weren't visible in lane 3

  • Dimers, trimers and etc were hardly visible maybe severely diluted

In lane 7 had detectable by very weak band

  • There weren't any problems with gel extraction