02/09/13

From 2013.igem.org

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(→Volatile organic compound (VOC) experiment of limonene biobrick/pBS1C3 backbone)
 
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* The x2 400ml agar were then placed in the 37 degrees room to cool.
* The x2 400ml agar were then placed in the 37 degrees room to cool.
* After cooling, 800ul of cholramphenicol and 400ul of IPTG were added to one bottle whilst only 800ul of chloramphenicol was added to the second bottle.
* After cooling, 800ul of cholramphenicol and 400ul of IPTG were added to one bottle whilst only 800ul of chloramphenicol was added to the second bottle.
-
* The contents of both agar bottles was then poured into ...
+
* The content of both agar bottles was then poured into petri dishes.  
 +
* 3 (chloramphenicol+ IPTG) plates were streaked with 5.1 ligated product (limonene biobrick and chloramphenicol backbone) from earlier and 3 others were streaked with 10.2 ligated product.
 +
* RFP colonies were streaked on 3 chloramphenicol resistance plates.
 +
* All 9 plates were Incubated overnight in a 37 degrees room.
 +
 
 +
==Ligation of PCR products (TOD operon) using the Promega pGEM-T Vector System==
 +
The samples ligated were A (TODX), C (TODF) and E (TOBG)
 +
 
 +
The promega protocol was followed and it can be found at http://www.promega.co.uk/resources/protocols/technical-manuals/0/pgem-t-and-pgem-t-easy-vector-systems-protocol/
 +
 
 +
To calculate the amount of DNA insert needed for each sample, we used the promega biomath calculator (www.promega.com/biomath). We used the 1:3 ratio of vector to insert.
 +
 
 +
*Size of the PCR inserts (add 56bp to account for the primer)
 +
**TodX 544 + 56= 600
 +
**TodF 460+56= 516
 +
**TobG 807 + 56=863
 +
 
 +
 
 +
*Volume of DNA insert for each sample
 +
**A - 1.6ul
 +
**C - 1.1ul
 +
**E - 1.5ul
 +
 
 +
The samples were incubate at 4 degrees overnight to be transformed tomorrow morning.
 +
 
 +
== Overnight Broth==
 +
* RFP cells were incubated in luria broth in a 37 degrees shaker incubator
 +
* The cells would be minipreped tomorrow to serve as a backbone for the ligation of the TOD biobbricks.
 +
 
 +
How to make the broth
 +
*Using a 15ml centrifuge tube add:
 +
**5ml of luria broth
 +
**5ul of Chloramphenicol
 +
 +
== Preparation of Ampicillin Resistance plates==
 +
* Ampicillin resistance plates were made by adding 400ul of Ampicillin to 400ml of agar.
 +
* This would be used to grow the transformants of samples A,C and E (above) as pGEM-T Vector has AmpR gene.
 +
 
 +
==Volatile organic compound (VOC) experiment of limonene biobrick/pBS1C3 backbone==
 +
* 9 plates were taken over to our chemistry department to measure the expression of limonene.
 +
* The equipment used for this assay is a Proton Transfer Reaction- Time of Flight Mass Spectrometer.
 +
 
 +
PLATES
 +
* 3 plates of 5.1(see [[05/08/13]]) ligated product
 +
* 3 plates of 10.2 (see [[05/08/13]])ligated product
 +
* 3 luria agar plates ( to be used as control, for blanking the equipment)

Latest revision as of 15:10, 12 September 2013

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Contents

Preparation of IPTG and Chloramphenicol plates

  • The IPTG/Chloramphenicol plates that were prepared earlier on (08/08/13) for the volatile organic compound (VOC) test were found to be contaminated.
  • Hence new plates had to be made using the below protocol;

PROTOCOL:

  • x2 400ml of already prepared and sterilized agar was obtained from the media kitchen.
  • The agar was microwaved using the lowest power level at 2-3 mins intervals until completely melted.
  • The x2 400ml agar were then placed in the 37 degrees room to cool.
  • After cooling, 800ul of cholramphenicol and 400ul of IPTG were added to one bottle whilst only 800ul of chloramphenicol was added to the second bottle.
  • The content of both agar bottles was then poured into petri dishes.
  • 3 (chloramphenicol+ IPTG) plates were streaked with 5.1 ligated product (limonene biobrick and chloramphenicol backbone) from earlier and 3 others were streaked with 10.2 ligated product.
  • RFP colonies were streaked on 3 chloramphenicol resistance plates.
  • All 9 plates were Incubated overnight in a 37 degrees room.

Ligation of PCR products (TOD operon) using the Promega pGEM-T Vector System

The samples ligated were A (TODX), C (TODF) and E (TOBG)

The promega protocol was followed and it can be found at http://www.promega.co.uk/resources/protocols/technical-manuals/0/pgem-t-and-pgem-t-easy-vector-systems-protocol/

To calculate the amount of DNA insert needed for each sample, we used the promega biomath calculator (www.promega.com/biomath). We used the 1:3 ratio of vector to insert.

  • Size of the PCR inserts (add 56bp to account for the primer)
    • TodX 544 + 56= 600
    • TodF 460+56= 516
    • TobG 807 + 56=863


  • Volume of DNA insert for each sample
    • A - 1.6ul
    • C - 1.1ul
    • E - 1.5ul

The samples were incubate at 4 degrees overnight to be transformed tomorrow morning.

Overnight Broth

  • RFP cells were incubated in luria broth in a 37 degrees shaker incubator
  • The cells would be minipreped tomorrow to serve as a backbone for the ligation of the TOD biobbricks.

How to make the broth

  • Using a 15ml centrifuge tube add:
    • 5ml of luria broth
    • 5ul of Chloramphenicol

Preparation of Ampicillin Resistance plates

  • Ampicillin resistance plates were made by adding 400ul of Ampicillin to 400ml of agar.
  • This would be used to grow the transformants of samples A,C and E (above) as pGEM-T Vector has AmpR gene.

Volatile organic compound (VOC) experiment of limonene biobrick/pBS1C3 backbone

  • 9 plates were taken over to our chemistry department to measure the expression of limonene.
  • The equipment used for this assay is a Proton Transfer Reaction- Time of Flight Mass Spectrometer.

PLATES

  • 3 plates of 5.1(see 05/08/13) ligated product
  • 3 plates of 10.2 (see 05/08/13)ligated product
  • 3 luria agar plates ( to be used as control, for blanking the equipment)
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