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02/09/13
From 2013.igem.org
(Difference between revisions)
(→Ligation of PCR products (TOD operon) using the Promega pGEM-T Vector System) |
(→Preparation of IPTG and Chloramphenicol plates) |
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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 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== | ==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 samples ligated were A (TODX), C (TODF) and E (TOBG) | ||
Revision as of 16:06, 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 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.
