Team:Paris/August 7

Glycerol Stocks

• 1mL of each culture (with 2 clones) has been added to 1mL of 40% glycerol.
• For each clone, two glycerol stocks have been done.
• Stored at -20°C.

Result of the isolation of colonies

E0240 and pSB3K3

E0240 and pSB3K3 are ok : there is a lot of single colonies
Two colonies are picked for an overnight culture at 37°C, 225 rpm, in order to extract plasmids (MiniPreps)

S120 and S121

S120 and S121 : there is a problem, there is nothing on the plates. We have to check whether those strains are really resistant to Amp.
We checked in the strain library, actually the strains do not carry any resistance cassette. We plated them once again on petri dishes with LB without antibiotics

Preparation of the newly ammplified promoters

Electrophoresis of the PCR products made yesterday

Standart PCR to amplify pflgA, pflgB and pflhB(Gel1)

PCR with gradient to amplify pflhDC(Gel2)

Electrophoresis settings

• Gel : 1.5 % agar
• 3µL template DNA
• 10µL QuickLoad DNA ladder 100 bp

Results

• We have no results for pflhDC, wo don't know yet where is the problem. We will try with other conditions! (yet undetermined)
• Concerning pflhB, pflgA and pflgB, the protocol seems to be very operational: we always have great results !

Washing of the PCR products

• Kit used : Wizard SV Gel and PCR Clean-Up System from Promega
• Standart protocol except the last step. Instead of eluting with water, we used 30 µL of BE buffer (from Qiagen)

DNA concentration measurement

We used two methods:

With a Spectrophotometer

• λ = 260 nm
• White: 100µL BE Buffer
• Templates : 2 µL DNA + 98 µL BE Buffer

With a Biophotometer

Remarks :

• There is a great difference between the two methods : sometimes 1 log !
• The electrophoresis of the PCR products showed that pflgB was more concentrated than pflgA and pflhA. As a consequence, we rely on the figures determined by the Biophotometer

Digestion

Gel 1 - 1% agar, ladder 1kb

Gel 2 - 1.5 % agar, ladder 100bp

Protocol

Digestion mix:

• X µL of template DNA
• 1 µL Enzyme 1
• 1 µL Enzyme 2
• 3 µL Buffer P2
• 0.3 µL BSA
• 24.7 - X µL of distilled water

We pu the digestion mix to incubate during 2h at 37°C

Results

Transformations

Protocol

Use of TOP10 chemically competentcells

• Defroze competent cells on ice during 5'
• Add 5µl of DNA Ligation in 50µL of competent bacterias (or 1µL for the positive control puc19)
• Incubate 30' on ice
• Heat-shock the cells during 30" at 42°C without shaking
• Put 2' on ice
• Add 250µL of pre-warmed SOC medium (4°C)
• Incubate 1h at 37°C under shaking (225rpm)
• Spin at 5.000rpm during 30"
• Remove 150µL of supernatant
• Resuspent the pellet in the 150µL left
• Spread on adequated plates
• Incubate O/N at 37°C

List of the Ligation Transformation

PCR Screening of Ligation Transformants of 1st August

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
• 10µl of screening PCR
• migration ~30min at 100W on 1% gel

Results

PCR1_’’’L101(1-8)’’’			PCR2_’’’L102(1-8)’’’			PCR3_’’’L113(1-8)’’’			PCR4_’’’L114(1-8)’’’
Expected size	Measured size	Band	Expected size	Measured size	Band	Expected size	Measured size	Band	Expected size	Measured size	Band
		2-9			10-17			1, 3-9			10-17
Gel 1 : L100-L101						Gel 2 : L113-L114

PCR5_’’’L120(1-8)’’’			PCR6_’’’L122(1-4)’’’			PCR7_’’’L123(1-8)’’’			PCR8_’’’L126(1-6)’’’
Expected size	Measured size	Band	Expected size	Measured size	Band	Expected size	Measured size	Band	Expected size	Measured size	Band
		1,2, 4-9			10-13			2-8, 1			3-8
Gel 3 : L120-L122						Gel 4 : L123-L126

PCR5_’’’L126(7-8)’’’
Expected size	Measured size	Band
		2-3
Gel 5 : L126

==> Conclusion :