Azurinlabbrapport

Hur gör man Azurin?

And here we have it; copy paste fån min gammla rapport:

Sterile technique is used during the whole laboration.
Day 1
DNA extraction.
1) Pick a single bacteria colony from an agar-plate using a sterile toothpick.
2) Dip the toothpick in a small volume of sterile H2O (50μl) and suspend the bacteria.
3) Boil for 5 min at 98°C in heat block and collect the cell debris by centrifugation. Centrifugate for 5 min at 13000 rpm. Use the supernatant as the template for PCR reaction.

Megaprimer-method.
4 primers are used:
• AzurinE106G vs. Azurin down in first PCR: 171 bp
• Megaprimer vs. Azurin up in second PCR

Perform a PCR, this first PCR uses the 1st flanking primer and the mutation primer to construct a megaprimer.

PCR 1
a) Add following in a eppendorf tube:
5 μl 10X PCR Buffer
5 μl dNTPs (2mM)
2 μl AzurinE106G
2 μl Azurin down
1 μl Taq Polymeras (1u/1 μl keep cold!)
12 μl H2O
3 μl MgSO4
20 µl Template DNA
Total= 50 μl


1. To mix, carefully pipette a couple of times. Keep cold.
2. Program the PCR machine and start the PCR reaction. The machine should be programmed according to:
Initial denaturation step: 96°C for 1 minutes
Denaturation step: 96°C for 0.5 minutes
Primer annealing step: 52°C for 0.5 minutes
Extending step: 74°C for 1 minute
Number of cycles: 32
Final extending step: 74°C for 5 minutes
Keep on 4ºC ∞

Purificate the megaprimer by using QIAquick® PCR Purification Kit. For protocol; see appendix.

From the purified sample first take 20 μl, to an eppendorf tube, mark it and place it in the freezer. Keep the rest of the sample in another tube. This will be the "Megaprimer sample" which will be run on the gel during day 4.

Perform a second PCR which uses the megaprimer and the 2nd flanking primer, according to the following PCR protocol.

PCR 2
5 μl 10X PCR Buffer
5 μl dNTPs (2mM)
20 μl Megaprimer
2 μl AzurinDown
1 μl Taq Polymeras (1u/1 μl keep cold!))
3 μl MgSO4
14 µl Template DNA
Vtotal= 50 μl

1. To mix, carefully pipette a couple of times. Keep cold.
2. Program the PCR machine and start the PCR reaction. The machine should be programmed according to:
Initial denaturation step: 96°C for 1 minutes
Denaturation step: 96°C for 0.5 minutes
Primer annealing step: 52°C for 0.5 minutes
Extending step: 74°C for 1 minute
Number of cycles: 32
Final extending step: 74°C for 5 minutes
Keep on 4ºC ∞

Purificate the PCR product by using QIAquick® PCR Purification Kit. For protocol; see appendix.

From the purified sample first take 20 μl, to an eppendorf tube, mark it and place it in the freezer. Keep the rest of the sample in another tube This will be the "Insert sample" which will be run on the gel during day 4.

Restriction enzyme digestion.
The product from PCR 2 resp. vector PCR-Script Amp SK(+) are cleaved with the restriction enzymes EcoRI and XbaI.

1. Add the following to a PCR tube marked "insert":
18 μl purified PCR product
5 μl 10X Buffer 2x Y+Tango™
1 μl EcoRI
1 μl XbaI
Vtotal= 25 μl

2. To mix, carefully pipette a couple of times. Keep cold.
3. Add the following to another PCR tube marked "vector":
10 μl PCR-Script Amp SK(+)
4 μl 10X Buffer 2x Y+Tango™
1 μl EcoRI
1 μl XbaI
4 μl H2O
Vtotal= 20 μl

4. To mix, carefully pipette a couple of times. Keep cold.
5. Place the tubes in a PCR-block kept at 37°C for 1h. Leave the PCR machine on 4°C during night.

Prepare plates and media for DH5α and BL21(DE3) (see appendix).
Day 2
1) Purificate the restriction enzyme cleaving products by using QIAquick® PCR Purification Kit. For protocol see appendix.

Ligation
1. Add the following to a PCR tube:
2.5 μl 10x ligation buffer
15 μl digested PCR product
5 μl digested PCR-Script Amp SK(+) vector
0.5 μl T4 DNA ligase
2 μl H2O
Vtotal= 25 μl

2. Incubate the mixture at room temperature for 1 hour.
3. Inactivate T4 ligase by heating the tube on a heat block to 65°C for 10 min.

Transformation 1 (E. coli DH5 α)
1. Thaw the competent cells on ice.
2. Mix 100µl cells with all of the ligation mix.
3. Store on ice 30 minutes and snap the tube every 10 minutes to avoid sedimentation.
4. Heat chock at 42°C for 2 minutes followed by 10 minutes on ice.
5. Transfer the transformed cells to 2ml LB medium in a 15 ml falcon tube. Incubate in 37°C in a shaker for 30 minutes.
6. Centrifuge in the swingout rotor and resuspend the pellet in 100µl LB medium to decrease the volume. Spread out 80 µl cell solution with 120 µl LB-medium and 20 µl cell solution with 180 µl LB-medium on agar plates containing ampicillin, X-gal and IPTG. Incubate over night at 37°C.

Making the gel
Use protective nitrile gloves (blue) and UV protecting glasses.
1. Weigh out 0.7g of agarose into a 250mL conical flask. Add 110mL of 0.5xTBE , swirl to mix. It is good to use a large container, as long as it fits in the microwave, because the agarose boils over easily.
2. Microwave for about 1 minute to dissolve the agarose. The agarose solution can boil over very easily so keep checking it. It is good to stop it after 45 seconds and give it a swirl. It can become superheated and NOT boil until you take it out whereupon it boils out all over you hands. So wear gloves and hold it at arms length.
3. Leave it to cool on the bench for 5 minutes down to about 60°C (just too hot to keep holding in bare hands).
4. Add 1 µl ethidium bromide to the solution. Remember gloves and glasses. Pour the gel slowly into the tank. Push any bubbles away to the side using a disposable tip. Insert the comb and double check that it is correctly positioned. The benefit of pouring slowly is that most bubbles stay up in the flask. Rinse out the flask immediately.
5. Leave to set for approximately 30 minutes with the lid open.
6. When the gel is ready, cover it with 0.5xTBE buffer and the plastic cover and leave it until further usage in the fume hood.
Day 3
1. Add 5 ml of sterile LB-medium to a sterile 15 ml falcon tube.
2. Add 5 µl of ampicillin (100mg/ml)
3. Pick a single white colony with a sterile toothpick and dip it into the LBA-medium.
4. Incubate at 37°C in a shake incubator during night.
Day 4
1) Isolate the plasmid for both the transformation and the control-cut, by using QIAprep Spin Miniprep Kit. For protocol see appendix.

Control cut
1. Add the following to a PCR tube:
5 μl purified DNA
1.5 μl 10X Buffer Y+Tango™
1 μl ClaI
7.5 μl H2O
Vtotal= 15 μl

2. To mix, carefully pipette a couple of times. Keep cold.
3. Place in a PCR-block kept at 37°C for 1 hour.
4. From the sample first take 20 μl to an eppendorf tube, mark it and keep cold. This will be the "RE control cut sample" which will be run on the gel. Keep rest in another tube.

Transformation 2
Transform to competent expression E.coli strain BL21(DE3).
a) Thaw the competent BL21(DE3) cells on ice. They will loose their activity if they get too warm.
b) Mix 5 µl of the isolated plasmid with 100 µl competent cells.
c) Store on ice 30 minutes and snap the tube every 10 minutes to avoid sedimentation.
d) Heat chock at 42°C for 2 minutes followed by 10 minutes on ice. Transfer the transformed cells to 2ml LB medium in a 15 ml falcon tube. Incubate in 37°C in a shaker for 30 minutes.
e) Centrifuge in the swing out rotor and resuspend the pellet in 100µl LB medium to decrease the volume.
f) Spread out 80 µl cell solution with 120 µl LB-medium and 20 µl cell solution with 180 µl LB-medium on LBA agar plates. Incubate over night at 37°C.

Perform a Agarose gel electrophoresis according to the protocol in the appendix.

Gelelectrophorese

SAMPLES EXPECTED LENGTH DILUTION
SAMPLE Loading dye
Megaprimer 171 bp 10 µl 2 µl
Insert 553 bp 10 µl 2 µl
RE control cut ~ 539 bp + vector 10 µl 2 µl
PCR-Script Amp SK(+) vector 3.0 kb 10 µl 2 µl

1. Load 10µl of Megaprimer , Insert , RE control cut and PCR-Script Amp SK(+) vector into fresh tubes. Add 2µl loading buffer (6X Loading Dye Solution) to each tube.
2. Load the first well with 5 µl of DNA Ladder plus (see picture in appendix). Continue with loading the other wells with your samples.
3. Connect the cables and switch on the current. Run the gel at 100 V for 30 min and then at 130 V until the blue dye has reached the other end approx. 60 min.
4. Look at the gel on a UV-light table. Use protective glasses.


For ladder see appendix, fig3.
Day 5
2) Check plates for white colonies (10 min).

Härlig laborationsrapport antar jag? :p

Rapporten är inte svår den är till och med obefintlig labbhandledningen är klurigare. Tack lotta för svaret dock det kommer hjälpa, frågade mest på skoj när jag var för trött för att skriva.