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Team:TU Delft/14 July 2010 content
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The PCR products were run on gel a 1% agarose gel (10 ul sample + 2 ul LB) for 1h at 100V. | The PCR products were run on gel a 1% agarose gel (10 ul sample + 2 ul LB) for 1h at 100V. | ||
| - | [[Image:TU_Delft_2010-07-14_Colony_PCR.png|thumb|Colony PCR Products| | + | [[Image:TU_Delft_2010-07-14_Colony_PCR.png|thumb|Colony PCR Products|440px]] |
'''Lane Description''' | '''Lane Description''' | ||
Revision as of 06:08, 16 July 2010
Emulsifier
By Pieter
Today I picked the colonies that grew over night from the transformation of:
| Ligation | Content |
| L1 | Control (just the plasmid backbone pSB1T3) |
| L2 | R0011+B0032 |
| L3 | OprG + B0015 |
| L4 | AlnA + B0015 |
All plates contained red and white colonies. From the control plate I picked one red colony. This is probably due to the fact that we did not purified the backbone before ligation. So some plasmids reassembled.
From the other three plates I picked 5 colonies each. I tried to avoid the red ones.
The colonies were picked according to our protocol (@TODO add link to protocol) and Colony PCR was conducted. The cultures were set in the stove to grow over night at 37 C.
The PCR products were run on gel a 1% agarose gel (10 ul sample + 2 ul LB) for 1h at 100V.
Lane Description
| # | Description |
| 0 | SmartLadder |
| 1 | L2 sample 1 |
| 2 | L2 sample 2 |
| 3 | L2 sample 3 |
| 4 | L2 sample 4 |
| 5 | L2 sample 5 |
| 6 | L3 sample 1 |
| 7 | L3 sample 2 |
| 8 | L3 sample 3 |
| 9 | L3 sample 4 |
| 10 | L3 sample 5 |
| 11 | L4 sample 1 |
| 12 | L4 sample 2 |
| 13 | L4 sample 3 |
| 14 | L4 sample 4 |
| 15 | L4 sample 5 |
| 16 | L1 sample 1 |
L2 Expected length of R0011 + B0032 = 55 + 13 = 83 bp (without flanking primer regions). On gel there are only bands poorly visible at height of 1500 bp. So the transformation failed.
L3 Expected lenght of OprG + B0015 = 744 + 130 = 870 bp (without flanking primer regions). On gel bands are visible of about 1000 bp in lanes 6,7 and 10. The bands just a little lower are unidentified.
L4 Expected length of AlnA + B0015 = 1107 + 130 = 1237 bp (without flanking primer regions). On gel bands are visible of about 1400 bp in lanes 11,13 and 15.
L1 Expected length of J04450 is 1090 bp (without flanking primer regions). On gel one band is visible in lane 16 at height 1400 bp.
From this gel it is clear that the transformation of the Promotor and RBS has failed. The OprG with stopcodon is probably in cultures 6 and 7. Since bands of AlnA with stopcodon and J04450 are about the same size, it is not clear whether this transformation succeeded. Tomorrow I will check the isolated plasmids on gel.
