02/09/13

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Revision as of 15:31, 2 September 2013

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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 contents of both agar bottles was then poured into ...

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.

@@ Line 39: / Line 39: @@
 **C - 1.1ul
 **E - 1.ul
+The samples were incubate at 4 degrees overnight to be transformed tomorrow morning.