Team:Paris/July 29

DNA digestion and purification

for each reaction (total volume : 50 µL)

• 20 µL of DNA (MiniPrep product)
• 2 µL of enzyme 1
• 2 µL of enzyme 2
• 5 µL of buffer 2 (10X)
• 0,5 µL of BSA
• 20,5 µL water

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:

• 2 µL of DNA
• 3 µL of water
• 1 µL of 6X loading dye

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 reconducted tomorrow.

PCR Screening of the Ligation Transformants

Use of 8 clones of Ligation transformants for screening PCR

Protocol of screening PCR

• Mix

• 50µl of Mix PCR by tube/clone
• one toothpick of each clone's colony by tube
• Program : Annealing 55°C - Time élongation 1'30" - Number cycle : 29

Conditions of electrophoresis

• 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

PCR1_’’’L102(1-8)’’’			PCR2_’’’L103(1-8)’’’			PCR3_’’’L104(1-8)’’’			PCR4_’’’L105(1-8)’’’
Expected size	Measured size	Band	Expected size	Measured size	Band	Expected size	Measured size	Band	Expected size	Measured size	Band
1045 pb	1000 pb	2-->9	1003 pb	1000 pb	10-->17	1078 pb	1000pb	2-->9	1239 pb	1200 pb	10-->17
Gel 1 : L102-L103						Gel 2 : L104-L105

PCR5_’’’L106(1-8)’’’			PCR6_’’’L107(1-8)’’’			PCR7_’’’L108.1(1-8)’’’			PCR8_’’’L108.2(1-8)’’’
Expected size	Measured size	Band	Expected size	Measured size	Band	Expected size	Measured size	Band	Expected size	Measured size	Band
1200 pb	1200 pb	2-->9	1239 pb	1200 pb	10-->17	1200 pb	1200 pb	2-->9	1200 pb	1200 pb	10-->17
Gel 3 : L106-L107						Gel 4 : L108.1-L108.2

PCR9_’’’L110(1-8)’’’			PCR10_’’’L111(1-8)’’’			PCR11_’’’L112(1-8)’’’			PCR12_’’’L115(1-7)’’’
Expected size	Measured size	Band	Expected size	Measured size	Band	Expected size	Measured size	Band	Expected size	Measured size	Band
1200 pb	1100 pb	2-->9	1239 pb	1100 pb	10-->17	1200 pb	1000 pb	2-->10	1239 pb	700 pb	11-->16
Gel 5 : L110-L111						Gel 6 : L112-L115

PCR13_’’’L115(8)’’’			PCR14_’’’L116(1-8)’’’			PCR15_’’’L117(1-6)’’’
Expected size	Measured size	Band	Expected size	Measured size	Band	Expected size	Measured size	Band
1239 pb	1200 pb	2	1200 pb	1200 pb	3-->10	1046 pb	1000 pb	11-->16
Gel 7 : L115-L116-L117

PCR16_’’’L117(7-8)’’’			PCR17_’’’L118(1-8)’’’			PCR18_’’’L119(1-5)’’’
Expected size	Measured size	Band	Expected size	Measured size	Band	Expected size	Measured size	Band
1046 pb	1000 pb	2-->3	1004 pb	1100 pb	4-->11	1079 pb	1200 pb	12-->16
Gel 8 : L117-L118-L119

PCR19_’’’L119(6-8)’’’			PCR20_’’’L120(1-8)’’’			PCR21_’’’L121(1-4)’’’
Expected size	Measured size	Band	Expected size	Measured size	Band	Expected size	Measured size	Band
1079 pb	1100 pb	2-->4	1239 pb	1000 pb	5-->12	1200 pb	1200 pb	13-->16
Gel 9 : L119-L120-L121

PCR22_’’’L121(5-8)’’’			PCR23_’’’L122(1-8)’’’			PCR24_’’’L125(1-4)’’’
Expected size	Measured size	Band	Expected size	Measured size	Band	Expected size	Measured size	Band
1200 pb	1000 pb	2-->5	1239 pb	1000 pb	6-->12	1045 pb	1000pb	13-->16
Gel 10 : L121-L122-L125

PCR25_’’’L125(5-8)’’’			PCR26_’’’L109.1(1-7)’’’			PCR27_’’’L109.2(1-8)’’’
Expected size	Measured size	Band	Expected size	Measured size	Band	Expected size	Measured size	Band
1045 pb	1000 pb	2-->5	1239 pb	1100 pb	2-->9	1239 pb	1100 pb	10-->17
Gel 11 : L125			Gel 14 : L109.1-L109.2

==> Conclusion : with the PCR, we have check that the transformant bacteria contain insert. (obtain amplification at the good size).

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:

PCR1_’’’L102(3;6))’’’			PCR2_’’’L103(4)’’’			PCR5_’’’L106(1; 2; 4)’’’			PCR10_’’’L111(1)’’’
Expected size	Measured size	Band	Expected size	Measured size	Band	Expected size	Measured size	Band	Expected size	Measured size	Band
1045 pb	1000 pb	2-3	1003 pb	-	4	1200 pb	1100 pb	5-6-7	1239 pb	1100 pb	8
Gel 13 : to solve Mistakes

==> Conclusion : We can observe a results for the samples : L102, L103, L106(4) and L111. (but not for L106(1; 2))