04/09/13

From 2013.igem.org

(Difference between revisions)
(→Double digest of TOD gene ligations)
(→Gel purification of the pSB1C3 backbone and RFP biobrick)
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*The Zymoclean Gel DNA recovery Kit was used to perform the purification and its protocol was followed.
*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.
*However the elution step was changed to 12ul of elution buffer, and this step was repeated.
 +
 +
Nanodrop of the samples
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 +
{|border=1
 +
|Sample||Volume||Concentration ng/ul||260/280||260/230
 +
|-
 +
|pSB1C3 1||19.8||31.9||1.83||1.75
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|-
 +
|pSB1C3 2||20||34||1.67||0.92
 +
|-
 +
|RFP 1||20||28.8||1.57||0.71
 +
|-
 +
|RFP 2||18.8||22.2||1.79||1.84
 +
|}
==Double digest of TOD genes PCR products==
==Double digest of TOD genes PCR products==

Revision as of 09:14, 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.

Nanodrop of the samples

SampleVolumeConcentration ng/ul260/280260/230
pSB1C3 119.831.91.831.75
pSB1C3 220341.670.92
RFP 12028.81.570.71
RFP 218.822.21.791.84

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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