IGEM:UBC/2009/Notebook/UBC iGEM 2010/2010/06/10
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Making AMP Plates
=PCR Screening of Transformed Cell Colonies
1. Below is the code for each transformed cell colony. Ex: F1a F is the part type (fluorescent protein), 1 is the part number, and a is the colony location (noted on plate))
F1=part BBa_E0040 F2=BBa_E1010 F3=BBa_E0020 F4=E0030
C1=BBa_I74033 (Constitutive promoter) C2=BBa_I74044
T1=BBa_B1006 (Forward terminator) T2=BBa_B0011 (Bi directional terminator) T3=BBa_B00211 (Bi directional terminator)
See below for the recipe for the PCR reagents (inserting table later):
Name 1X Volume μL 35.2 X Volume (Master Mix) 10 μM VF2 1.25 44.0 10 μM VR 1.25 44.0 10 dNTP 0.50 17.6
2. The samples were put in the PCR machine with the following steps:
1. 95°C--10 min 2. 95°C--30 secs 3. 56°C--30 sec 4. 72°C--1 min 5. Repeat 1-4 30 times 6. 68°C--20 min 7. Hold at 4°C
Total time of the PCR reaction: 1 h 30 minutes.
3. Afterwards, 5 μL of DNA from the PCR reaction (from each colony) was mixed with 1 μL loading dye. A 100 bp ladder was diluted 3 μL in 20 μL total volume with sdH20. 5 μL was then mixed with 1 μL loading dye. The ladder and PCR reaction products (mixed with loading dye) were then loaded into a gel box and run for 45 minutes at 100 V. The gel contained 0.8% agarose in 0.5 X TBE. The electrophoresis buffer was also 0.5 X TBE.
The electrophoresis results after 45 minutes show expected banding for every part(showing the part should exist within the transformed colonies). It was expected that the F# parts should have a single band around 1 kb (compared to ladder). All other parts have a single band at 350-450 kb, which was visible.
The exception was F3 (BBa_E0020), where no bands were visible. Due to the presence of satellite colonies, F4 was not colony PCR'd.