Team:Paris/August 8

Minipreps: Plasmid extraction
Extraction of pSB3K3 et E0240 in pSB1A2 plasmid from overnight bacteria culture using the QIAspin Miniprep Kit (QIAGEN) by QIACube.


 * Carried out 2 times (2 tubes)



Amplification of Genes of interest (OmpR, EnvZ, FlhDC)
We performed PCR on to amplify the sequence in order to have enough amount of DNA to carry out the following of our experiments.

Protocol

 * List of Oligos :


 * Preparation of the templates : Resuspension of 1 colony in 100µl of water.

For each sample,
 * Preparation of PCR mix :

1 µl dNTP 10 µl Buffer Phusion 5x 2,5 µl Oligo_F 2,5 µl Oligo_R 1µl template 1 µl Phusion 50 µl qsp H2O (33µl)




 * Program PCR_Screening : Annealing 55°C - Time élongation 1'30" - Number cycle : 29

PCR verification/Analysis
After the PCR :
 * 3µl have been analysed by electrophoresis
 * the other 47µl of PCR products have been purified by the Promega kit.


 * Electrophoresis

ladder : 10µl ladder 1 kb samples : 3µl of PCR products + 2µl of Loading Dye migration 30min at 100V, on a 1% agarose gel


 * Results :

==> Conclusion : we observed the size expected for the PCR products, but not for FlhDC gene. We hypothesis that its value of amplification for this gene it's low as we can't visualize it. So we try to continue to experiments to know if there is something inside or not this tube.


 * Quantification of the PCR products purified

Blank : 2µl of buffer EB + 98µl of water Samples : 2µl of PCR purified + 98µl of water.


 * Results :

Protocol :

 * Incubate 2h30 at 37°C

Analysis by electrophoresis

 * Conditions

ladder : 10µl ladder 1 kb samples : 3µl of insert + 2µl of Loading Dye migration 30min at 100V, on a 1% agarose gel.


 * Results :

==> Conclusions : We validate the digestion of the vector and the insert. Now we are sure, that we don't detect anything for FlhDC genes.

The return of the promoters
Yesterday, we could not see anything on the electrophoresis after the digestion. Today, we will try the digestion again.

Results
To analyze our digestions, we made an electrophoresis.
 * Gel : 1.5% Agar
 * Ladder : 100 bp
 * 4µL DNA + 2µL Loading Dye

Conclusion *The bands are not visible on this picture, but with a long exposition time, we managed to see the bands at their right place. *The concentration of DNA is low!

We will do the ligation tomorrow.

PCR Screening of Ligation Transformants
Use of 8 clones of Ligation transformants for PCR screening

Protocol of screening PCR

 * Mix

25µl of Quick Load 2X 1µl of forward primer 10µM 1µl of reverse primer 10µM 23µl of water


 * primers used


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

10µl of ladder 100 pb 10µl of screening PCR migration ~30min at 100V on 1,5% gel
 * Conditions of electrophoresis 

Results of electrophoresis
gel 1 gel 2

==> Conclusion:
 * PCR of pFlgA, pFlgB and pFlhB have succeed, but we always have a problem with pFlhDC probably because of the primers which are not specific.

Building of the standard measurement plasmid
This morning we MiniPreped E0240 and pSB3K3 which are the two important parts of the standard measurement plasmid.

Before the digestion, we have to determine the DNA concentration of the MiniPreps

Protocol

 * X µL of Template DNA
 * Buffer (n°2) 10X : 3µL
 * BSA 100X : 0.3µL
 * Pure water qsp 30 µL
 * 1 µL of each enzyme


 * Incubate during about 3h at 37°C, then 20 minutes at 65°C (to inactivate the enzymes).

Results of the digestion


Electrophoresis settings
 * Gel : 1 % agar
 * 4µL template DNA for D137
 * All the digestion product for D138 because we have to separate two products, the backbone measures 2056 bp and the insert we want to extract is 899 bp long.
 * 10µL QuickLoad DNA ladder 1 kb

Gel extraction and DNA purification
To extract the biobrick E0240 from the gel, we used the standard protocol number 8. To purify the DNA we used the standard protocol number 10

The ligation will be done tomorrow.

Protocol
PCR Mix for cloning with Taq polymerase
 * 25µL Quick-load Mix
 * 1µL Oligo F (10µM)
 * 1µL Oligo R (10µM)
 * 1µL Template DNA (MP 143)
 * 23µL pure water

We try to build a measurement tool with and without included RBS.

PCR Program LID 105°C 1. 95°C  5 min 2. 95°C  1 min 3. 60°C  30 sec 4. 72°C  1 min 30 5. go to : 2  rep : 29 6. sound : 1 7. hold : 10°C

Results
Electrophoresis settings
 * Gel : 1 % agar
 * 5µL PCR products
 * 10µL QuickLoad DNA ladder 1 kb

Conclusion: - There is DNA that is around 3,000 bp. Actually, it is the template DNA (MP 143), there were too much DNA. We will have to extract on gel the right piece of DNA. - RBS - worked better than RBS +.

Gel Extraction and DNA purification
Electrophoresis settings
 * Gel : 1 % agar
 * All the PCR products (Two bands for each template)
 * 10µL QuickLoad DNA ladder 1 kb

Once again we see that the PCR for RBS - worked a lot better than the PCR for RBS +. We will do it again on monday.

To extract RBS - from the gel, we used the standard protocol number 8. To purify the DNA we used the standard protocol number 10