Team:Paris/August 7

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Contents

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.
name Strain
S141 MG1655
S142 J61002


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 amplified 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
Name Promotor Gel Band Expected size Measured size
PCR_124 pFlgA 1 2
261 pb
250 pb
PCR_125 pFlgB 1 3
261 pb
250 pb
PCR_126 pFlhB 1 4
260 pb
250 pb
PCR_127 pFlhDC 2 2 to 13
446 pb
nothing




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


Template Absorbance Estimated
Concentration
(µg/µL)
pflgA 0.202 0.5
pflgB 0.210 0.5
pflhA 0.193 0.5

With a Biophotometer

Template Estimated
Concentration
(µg/µL)
Ratio DO260/DO280
pflgA 0.05 1.06
pflgB 0.1 1.44
pflhA 0.O5 1.66

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

Protocol

Gel 1 - 1% agar, ladder 1kb
Gel 2 - 1.5 % agar, ladder 100bp
Digestion name Template DNA Enzymes Quantity of DNA used
D132 PCR 124 - pflgA EcoRI-SpeI 2 µL
D133 PCR 125 - pflgB EcoRI-SpeI 1 µL
D134 PCR 16 - pflhB EcoRI-SpeI 2 µL
D136 MP123 EcoRI-SpeI 8 µL

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

Name Gel Band Expected size Measured size
D136 1 2
2925 pb
3000 pb
MP 123 1 4
3002 pb
3000 pb
D133 2 2
225 pb
nothing
D134 2 3
225 pb
nothing
D135 2 4
226 pb
nothing


    Conclusion : The digestion of MP123 into D136 worked very well. Tomorrow we will isolate it.
    We see nothing on the other digestion. Maybe someone forgot to put the template DNA inside the tube !? 
    Anyway, we will do the manipulation again tomorrow.

Transformations

Protocol

Use of TOP10 chemically competent cells

  • Defroze competent cells on ice during 5'
  • Add 5µl of Ligation products in 50µL of competent bacteria (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
  • Resuspend the pellet in the 150µL left
  • Spread on adequate plates
  • Incubate O/N at 37°C

List of the Ligation Transformation

Name Description Antibio
Ligation
L128 J61002-pFlgA
D136 (FV) - D132 (FI)
Amp
L129 J61002-pFlgB
D136 (FV) - D133 (FI)
Amp
L130 J61002-pFlhB
D136 (FV) - D134 (FI)
Amp
L131 J61002-pFlhDC
D136 (FV) - D135 (FI)
Amp
Control
Control 1 D136 Amp
Positive control pUC19 Amp


PCR Screening of Ligation Transformants of 1st August

Use of 8 clones of Ligation transformants for screening PCR


Protocol of screening PCR

  • Mix
Name Vol (µl) Concentration
Quick Load 25µl 2X
OligoF_VF2 (O18) 1µl 10µM
OligoR_VR (O19) 1µl 10µM
water 23µl


  • 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

Gel 1 : L100-L101KR000118 gel1.jpg Gel 2 : L113-L114KR000119 gel2.jpg Gel 3 : L120-L122KR000120 gel3.jpg
Gel 4 : L123-L126KR000122 gel4.jpg Gel 5 : L126KR000123 gel5.jpg

name Description Expected size Measured size Gel Band Comments
PCR1_’’’L101(1-8)’’’ D110-D131 1200 1100 1 2-9 (1-8) ok
PCR2_’’’L102(1-8)’’’ D129-D118 1045 1000 1 10-17 (1-8) ok
PCR3_’’’L113(1-8)’’’ D126-D130 1239 2500 2 1, 3-9 to do again
PCR4_’’’L114(1-8)’’’ D126-D131 1239 1200 2 10-17 (1-8) ok
PCR5_’’’L120(1-8)’’’ D106-D130 1239 2000 3 1,2, 4-9 (1-8) ok
PCR6_’’’L122(1-4)’’’ D107-D130 1239 1200 3 10-13 (1) ok
PCR7_’’’L123(1-8)’’’ D107-D131 1200 1100 4 & 4' 2-8, 1 (1-8) ok
PCR8_’’’L126(1-6)’’’ D102-D118 1045 1000 4' 3-8 (1-8) ok
PCR5_’’’L126(7-8)’’’ 1000 6 2-3


==> Conclusion :In futur , we will prepare minipreps and stocks for the transformant clones succesfull.