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02/09/13
From 2013.igem.org
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The samples were incubate at 4 degrees overnight to be transformed tomorrow morning. | 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. | ||
Revision as of 16:25, 2 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 contents 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.
