04/09/13

From 2013.igem.org

(Difference between revisions)
(→Gel Electrophoresis)
(→Double digest of TOD gene ligations)
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*However the elution step was changed to 12ul of elution buffer, and this step was repeated.
*However the elution step was changed to 12ul of elution buffer, and this step was repeated.
-
==Double digest of TOD gene ligations==
+
==Double digest of TOD genes PCR products==
 +
Samples A (TodX), C (TodF) and E (ToBG) were digested with the enzymes XbaI and SpeI.
 +
*Protocol
 +
{| border=1
 +
|Samples || A ||C ||E
 +
|-
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|Volume (ul)||20|| 23.5||21
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|-
 +
|SpeI (ul)||1|| 1|| 1
 +
|-
 +
|XbaI|| 0.5|| 0.5||0.5
 +
|-
 +
|Cutsmart Buffer|| 6|| 6||6
 +
|-
 +
|5mTris Hcl (ul)|| 32.5|| 29|| 31.5 
 +
|}
 +
 
 +
**The samples were incubated at 37C for 90 minutes
 +
**Heat kill the enzymes by incubating the samples at 80C for 20 minutes

Revision as of 09:07, 5 September 2013

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Contents

Transformation results

Gel Electrophoresis

  • The double digest (pSB1C3) from yesterday was run on a 1% gel.
  • This was done in order to confirm that the digest worked and also to purify the pSB1C3 vector.

Igem psb1c3 doubledigest 040913.jpg

  • From left to right
  1. Sample 1.1 - track 2
  2. Sample 1.2 - track 3
  3. Sample 2.1 - track 4
  4. Sample 2.2 - track 5
    • The 2 Kb bands are the pSB1C3 backbone
    • The 1 Kb bands are the RFP biobrick 
The two higher bands in tracks 2 and 3 are plasmids that were not digested.

Gel purification of the pSB1C3 backbone and RFP biobrick

  • The Zymoclean Gel DNA recovery Kit was used to perform the purification and its protocol was followed.
  • However the elution step was changed to 12ul of elution buffer, and this step was repeated.

Double digest of TOD genes PCR products

Samples A (TodX), C (TodF) and E (ToBG) were digested with the enzymes XbaI and SpeI.

  • Protocol
Samples A C E
Volume (ul)20 23.521
SpeI (ul)1 1 1
XbaI 0.5 0.50.5
Cutsmart Buffer 6 66
5mTris Hcl (ul) 32.5 29 31.5
    • The samples were incubated at 37C for 90 minutes
    • Heat kill the enzymes by incubating the samples at 80C for 20 minutes
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