Team:Paris/July 29
From 2008.igem.org
(→DNA digestion and purification) |
(→Screening PCR of the transformations with Ligation) |
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- | ===Protocol=== | + | ===Protocol of screening PCR=== |
+ | |||
* '''Mix''' | * '''Mix''' | ||
{| Border="1" | {| Border="1" | ||
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* one toothpick of each clone's colony by tube | * one toothpick of each clone's colony by tube | ||
* Program : Annealing 55°C - Time élongation 1'30" - Number cycle : 29 | * Program : Annealing 55°C - Time élongation 1'30" - Number cycle : 29 | ||
+ | |||
+ | |||
+ | === Conditions of migration === | ||
+ | |||
+ | |||
+ | * 10µl of ladder 1 kb | ||
+ | * 15µl of screening PCR (gel n°1, 2, 3(9-17), 4, 5, 6, 7, 8, 9, 10, 11) | ||
+ | * 10µl of screening PCR (gel n°3(1), 13, 14) | ||
+ | * migration ~30min at 100W on '''0,8%''' gel | ||
+ | |||
===Results=== | ===Results=== | ||
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{| border="1" | {| border="1" | ||
|colspan="3"|PCR25_’’’L125(5-8)’’’ | |colspan="3"|PCR25_’’’L125(5-8)’’’ | ||
- | |colspan="3"| | + | |colspan="3"|PCR26_’’’L109.1(1-7)’’’ |
|colspan="3"|PCR27_’’’L109.2(1-8)’’’ | |colspan="3"|PCR27_’’’L109.2(1-8)’’’ | ||
|- | |- | ||
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|colspan="6"|[[Image: KR000099_14.jpg|thumb|'''Gel 14 : L109.1-L109.2''']] | |colspan="6"|[[Image: KR000099_14.jpg|thumb|'''Gel 14 : L109.1-L109.2''']] | ||
|} | |} | ||
+ | |||
+ | |||
+ | ==> '''Conclusion :''' We have ..... | ||
+ | |||
+ | But we don't observe results for L102(3), L102(6), L103(4), L106(1), L106(2), L106(4), L111(1) | ||
+ | |||
+ | Migration of an another gel for this sample... | ||
+ | |||
+ | |||
+ | '''Results''': | ||
+ | |||
+ | * | ||
+ | {| border="1" | ||
+ | |colspan="3"|PCR1_’’’L102(3;6))’’’ | ||
+ | |colspan="3"|PCR2_’’’L103(4)’’’ | ||
+ | |colspan="3"|PCR5_’’’L106(1; 2; 4)’’’ | ||
+ | |colspan="3"|PCR10_’’’L111(1)’’’ | ||
+ | |||
+ | |- | ||
+ | |align="center"|'''Expected size''' | ||
+ | |align="center"|'''Measured size''' | ||
+ | |align="center"|'''Band''' | ||
+ | |align="center"|'''Expected size''' | ||
+ | |align="center"|'''Measured size''' | ||
+ | |align="center"|'''Band''' | ||
+ | |align="center"|'''Expected size''' | ||
+ | |align="center"|'''Measured size''' | ||
+ | |align="center"|'''Band''' | ||
+ | |align="center"|'''Expected size''' | ||
+ | |align="center"|'''Measured size''' | ||
+ | |align="center"|'''Band''' | ||
+ | |- | ||
+ | |align="center"| | ||
+ | |align="center"| | ||
+ | |align="center"|2-3 | ||
+ | |align="center"| | ||
+ | |align="center"| | ||
+ | |align="center"|4 | ||
+ | |align="center"| | ||
+ | |align="center"| | ||
+ | |align="center"|5-6-7 | ||
+ | |align="center"| | ||
+ | |align="center"| | ||
+ | |align="center"|8 | ||
+ | |- | ||
+ | |colspan="6"|[[Image: KR000098_13.jpg|thumb|'''Gel 13 : Mistakes''']] | ||
+ | |} | ||
+ | |||
+ | |||
+ | ==> '''Conclusion :''' We can observe a results for the samples : L102, L103, L106(4) and L111. | ||
+ | (but not for L106(1; 2) |
Revision as of 13:42, 31 July 2008
DNA digestion and purificationfor each reaction (total volume : 50 µL)
Each reaction was incubated 2 hours at 37°C, then 10 minutes at 60-65°C (to inactivate the enzymes). 10 µL of loading dye (6X) were added to each of the 50 µL of digestion product. The whole samples were run in a 1,5% agarose gel (about 30 minutes at 100 W ; 2 x 30 µL per sample ; 30 µL per well). The bands of interest were then excised from the gel and the DNA was purified using the QIAquick DNA Gel Extraction kit (QIAGEN). Unfortunately, there were not enough columms, so we took some columms from the QIAGEN MiniPrep kit, hoping that it will work with the QIAquick DNA Gel Extraction kit. Some of the samples were too voluminous, so we separated them into two tubes. The elution of DNA was performed using 50 µL of water (after 10 minutes of incubation at 37°C). Each of the samples was then analysed by a 1,5% agarose gel:
The ladder used was the 100 bp ladder from New England Biolabs.
Results The amount of DNA loaded was too low and the DNA ladder used was the wrong one. So this experiment has to be done one more time tomorrow. PCR Screening of the Ligation TransformantsUse of the same 8 clones of Biobricks that have been tested for cloning
Protocol of screening PCR
Conditions of migration
Results
But we don't observe results for L102(3), L102(6), L103(4), L106(1), L106(2), L106(4), L111(1) Migration of an another gel for this sample...
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