Team:Paris/Notebook/Protocols

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Revision as of 13:08, 11 August 2008

Culture of Stable strain with biobricks 2008

In 6ml LB with adaptated antibiotics
Will be use for Miniprep and Stock in glycerol
2 clones isolated by Biobricks
O/N at 37°C

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.

Minipreps (Kit Qiagen)

Resuspend pelleted bacterial cells in 250 µl Buffer P1 and transfer to a microcentrifuge tube.
Add 250 µl Buffer P2 and mix thoroughly by inverting the tube 4–6 times. If using LyseBlue reagent, solution turns blue.
Add 350 µl Buffer N3 and mix immediately and thoroughly by inverting the tube 4–6 times. If using LyseBlue reagent, solution turns colorless.
Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge.
Apply the supernatant (from step 4) to the QIAprep spin column by decanting or pipetting.
Centrifuge for 30–60 s. Discard the flow-through.
Recommended: Wash the QIAprep spin column by adding 0.5 ml Buffer PB and centrifuging for 30–60 s. Discard the flow-through. This step is only required when using endA+ or other bacteria strains with high nuclease activity or carbohydrate content (see QIAprep Miniprep Handbookfor more details)
Wash QIAprep spin column by adding 0.75 ml Buffer PE and centrifuging for 30–60 s.
Discard the flow-through, and centrifuge for an additional 1 min to remove residual wash buffer.
To elute DNA, place the QIAprep column in a clean 1.5 ml microcentrifuge tube. Add 50 µl Buffer EB or water to the center of each QIAprep spin column, let stand for 1 min, and centrifuge for 1 min.

Qualitative and quantitative analysis by electrophoresis

Gel : 1% agarose
10 µL Quick-Load 1 kb DNA Ladder
2 µL LB + 3 µL DNA
Bath of BET 20000X (5 µL BET for 100 mL TBE) during about 5 min.

=> Comparing the concentration of the miniprep thanks to the ladder
Check if the mesured size corresponds with the expecting size.

Concentration of the Miniprep

By biophotometry

Blank : 60 µL of pure water
Sample : 50 µL of pure water + 5 µLof DNA

Check if the ratio 260/280 is over 1,6
!!!Think about the dilution!!!

Amplification of promoters

=>To amplify the sequence in order to have enough amount of DNA to carry out the following of our experiments.

Preparation of the templates : Resuspend of 1 colony in 100µl of water.
Preparation of PCR mix :

For each samples, 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 : PROMOTEU

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

Digestion

1 µg of plasmid (Miniprep)
Buffer (n°2) 10X
BSA 100X
Pure water qsp 30 µL
1 µL of each enzyme

Incubate during about 3h at 37°C, then 10 minutes at 60-65°C (to inactivate the enzymes).

Migration after digestion

Separation of insert and vector

Run the whole samples in a 1,5% agarose gel
About 30 minutes at 100 W
10 µL of ladder 1kb and 100 pb on every side
3 µL of DNA + 2 µL of LB

!!!Separate each band by an empty one!!!

For promoters

1,5% agarose gel
10 µL of ladder 100 pb
3 µL of digestion product
2 µL of LB
Run at 100 W about 30 min

For vectors

1% agarose gel
10 µL of ladder 1 kb
3 µL of digestion product
2 µL of LB
Run at 50 W about 30 min

Extraction

For each new extraction it's important to have a new bath of BET
Use a new blade for each extraction
The extraction must be under 400 mg

@@ Line 48: / Line 48: @@
 * '''Preparation of PCR mix''' :
 ''For each samples,''
 µl dNTP
 <br>10 µl Buffer Phusion 5x
@@ Line 58: / Line 56: @@
 <br>50 µl qsp H2O (33µl)
-*Program PCR : PROMOTEU
+*Program PCR : '''PROMOTEU'''
 LID : 105°C<br>
 . 95°C   5 min<br>
@@ Line 66: / Line 64: @@
 . go to : 2   rep : 29
 . sound : 1
 . hold : 10°C
 ==Digestion==