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From 2013.igem.org

Contents 1 Preparation of IPTG and Chloramphenicol plates 2 Ligation of PCR products (TOD operon) using the Promega pGEM-T Vector System 3 Overnight Broth 4 Preparation of Ampicillin Resistance plates

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 from earlier and 3 others were streaked with 10.2 ligated products.
• 3 RFP plates were streaked on Chloramphenicol plates.
• All 9 plates were Incubated in 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.ul

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

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

Preparation of Ampicillin Resistance plates

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