Team:METU Turkey/protocols.html

4. GETTING THE DNA PARTS FROM KIT PLATE:

• 10 ul ddHO is added into the kit plate target part.

• Wait 5 min.and get the part by well pipetting.

 

5. TRANSFORMATION:

Materials:

• Resuspended DNA (2 ul )

• competent cells (50ul per transformation)

• Ice

• 2ml tube (1 per a transformation')

• 42ºC water bath

• Petri dishes with LB agar and appropriate antibiotic (2 or 3 per transformation)

• Glass beads or spreader

• 37ºC incubator

 

Preparation:

• Start thawing the competent cells on ice.

• Add 50 µL of thawed competent cells into pre¬chilled 2ml tube.

• Add 1 - 2 µL of the resuspended DNA to the 2ml tube. Pipet up and down a few times,

gently. Make sure to keep the competent cells on ice.

• Close tube and incubate the cells on ice for 30 minutes.

• Heat shock the cells by immersion in a pre-heated water bath at 42ºC for 60 seconds.• Incubate the cells on ice for 5 minutes.

• Add 400 µl of LB media (make sure that the broth does not contain antibiotics and is not

contaminated)

• Incubate the cells at 37ºC for 2 hours while the tubes are rotating or shaking. Important: 2

hour recovery time helps in transformation efficiency, especially for plasmid backbones with

antibiotic resistance other than ampicillin.

• Label two petri dishes with LB agar and the appropriate antibiotic(s) with the part number,

plasmid backbone, and antibiotic resistance. Plate 20 µl and 200 µl of the transformation onto the

dishes, and spread. This helps ensure that you will be able to pick out a single colony.

• Incubate the plate at 37ºC for 12-14 hours, making sure the agar side of the plate is up. If

incubated for too long the antibiotics start to break down and un-transformed cells will begin to

grow. This is especially true for ampicillin - because the resistance enzyme is excreted by the

bacteria, and inactivates the antibiotic outside of the bacteria.

• You can pick a single colony, make a glycerol stock, grow up a cell culture

 

Liquid Culture:

• 5 ml LB is mixed with appropriate antibiotic and a culture tube is prepared with certain

labels.

• A pipette tip is used to pick up a colony from the plate and release it into the LB by stirring

and pipetting up and down

• Incubate the culture at 37°C for 13-15 hours. Do not let the cells become old to not release

their plasmids

• If the culture becomes saturated: you can reinoculate 30µL into a new 3mL LB tube

 

Glycerol Stock:

• Put 500µL of a mid-log culture into a 1.5mL tube with 500µL 80% glycerol

• Store at ¬80C

Spreading Plates:

• Put 50 µL and 150 µL of whether LB culture or transformed cells.

• Spread the cells into the plate until there is no seen liquid.

• Wait until the plates dried.

• After 14¬16 hours colonies will show up.

 

Streaking Plates:

• Put a drop your culture on the plate

• Using a pipette tip, spread the drop out into a zigzag. Then use one edge of the zigzag to

draw out another zigzag. Repeat to have about 3 zigzags (this makes the culture get spread out

more and more with each streak)

• Wait until the plates dried.• After 14-16 hours colonies will show up.

 

Plasmid Isolation:

Usually there is a protocol in the kit. First, you should read it.

Take 2 ml cell in to the eppendorf (2ml).

Spin down 6 ml from the culture to the eppendorf at 8000rpm for 2 min.

Discard supernatant.

Add 2 ml cell in to the eppendorf again, totally 4 ml will be pelleted.

Spin down the cells at 8000rpm for 2 min.

Discard supernatant.

Resuspend the cells with 250 μl  with resuspension buffer (cold +4) and vortex.

Add 250 μl lysis buffer and gently inverse the eppendorf for 4-¬5 times. DON’T VORTEX!!!

Add 350 μl neutralisation solution and inverse gently and immediately.

Spin down the cells at 13000 rpm for 5 min.

Place the supernatant into the tube that is placed into the kit.

Spin down the cells at 13000 rpm for 1 min.

Remove the below part of the tube and discard supernatant.

Add 500 μl Wash buffer and santrifuge at 13000rpm for 1 min.

Add 500 μl  Wash buffer and santrifuge at 13000rpm for 1 min.

Spin down the cells at 13000rpm for 1 min.

Put the filter parts onto the clean 1.5 ml eppendorfs.

Add 50 μL elution buffer or 25 ul molecular gradient water.

Wait 2 min near the flame.

Spin down the cells at 13000rpm for 2 min.

Store your plasmids at +4C for 1 hour before further experiments or store at -20C if you don’t use them immediately.

 

 

 

1.1.Colony PCR:

Prepare mastermix according to your sample amount (except Taq and template)

(For 1x sample preparation)

• dNTP 1 µL

• Buffer 5 µL

• MgCl2 3 µL

• **Forward Primer 0,5 µL

• **Reverse Primer 0,5 µL

• Template ¬

• Taq Polymerase 0,2 µL

• dH2O 39,8 µL

• TOTAL 50 µL

* You should add ingredients from largest amount to smallest amount.

* Before addition of primers and template you can do vortex.

** First you should pour ddH20 onto dried primers according to the amount written in the primer

sheet then you should dilute (1:10) it in to new eppendorf. (10 ml primer+ 90 ml ddH20)

Pour your samples into PCR tubes and add template that you pick from the petri with toothpick or

tip and finally add taq polymerase.

Then place your samples into the PCR machine and do regular PCR.

1.2 PCR:

For a 25ul rxn:

• Use 1ul of 60ng/ul or 100ng/ul DNA

• Use 1ul of each primer at 3.2pmole/ul concentration or 1.25ul of each primer

at 100ng/ul concentration

• 2.5ul 10x PCR Buffer w/ Mg (1.5mM)

• 0.5ul 25mM MgCl2

• 0.5ul dNTP

• 0.125ul Taq

• 18.375ul sterile water to equal a 25ul rxn

 

(*if not making master mix, dilute Taq so that you can add 1ul of Taq and 17.5ul

sterile water to equal a 25ul rxn)

 

For a 50ul rxn:

 

• Use 2ul of 60ng/ul or 100ng/ul DNA

• Use 2ul of each primer at 3.2pmole/ul concentration or 2.5ul of each primer at

100ng/ul concentration

• 5ul 10x PCR Buffer w/ Mg

• 1ul 25mM MgCl2

• 1ul dNTP

• 0.25ul Taq

• 36.75ul sterile water to equal a 50ul rxn

(*if not making a master mix, dilute Taq so that you can add 1ul of Taq and 36ul

sterile water to equal a 50ul rxn)

 

2. Keep the reagents on ice.

3. Add the Taq last, and keep it in the freezer until you are ready to add it.

4. Vortex briefly and quick spin.

5. Cycle:

• 95°C for 1¬5minutes (usually 4min)

• 95°C for 1min

• 55°C for 1min

• Cycle 30 times

• 72°C for 1.5 to 2min (usually 2min)

• 72°C for 10min

• 4°C hold

 

 

 

6.RESTRICTION DIGESTION:

NEB Buffer 5 µl

BSA 0.5 µl

Enzyme 1 0.5 µl

Enzyme 2 0.5 µl

Plasmid*  1000ng/ml

To complete to 50 µl, add ddH2O.

1¬ Keep it in water bath for 2.5 hours at 37°C.

2¬ Run the samples on the gel.

7.LIGATION:

• 10X T4 ligase buffer: 2.0 µL

• 6:1 molar ratio of insert to vector (~10ng vector)

• Add (8.5 ¬ vector and insert volume)µl ddH2O

• T4 Ligase: 1 µL

• Incubation at room temperature 1 hour and 15 min at 65°C for enzyme inactivation.

• Alternatively: Incubation at +4°C overnight.

 

 

PCR Purification

We used the protocol provided with the QIAGEN kit.

• 3 volumes of Buffer QG is added to 1 volume of the PCR sample and mixed by vortexing.

• 1 volume of the INITIAL sample volume of isopropanol is added to the sample and mix.

• Place a QIAquick spin column in a provided 2 ml collection tube. Apply the sample to the

QIAquick column with the pipette and centrifuge for 30–60 s.Discard flow¬through. Place the

QIAquick column back into the same tube

• Add 0.75 ml Buffer PE to the QIAquick column with the pipette and centrifuge for 30–60 s.

Discard flow-through and place the QIAquick column back in the same tube.

• Centrifuge the column for an additional 1 min.

• Label the top of clean 1.7 ml microcentrifuge tube(s) with the name of your sample(s).

Transfer QIAquick column(s) to the tube(s).

• To elute DNA, add 50 µl Buffer EB (10 mM Tris∙Cl, pH 8.5) to the center of the QIAquick

membrane. Let it stand for 1 min and centrifuge the column for 1 min.

 

 

8.3 LB Medium

For 1 L of LB medium you need:

• 10 g Trypton

• 5 g yeast extract

• 10 g NaCl

• 12 g Agar¬Agar (for plates)

• Adjust pH to 7.0

8.4 LB Agar

For 1 L of LB Agar you need

• 10 g Trypton

• 5 g yeast extract

• 10 g NaCl

• 12 g Agar¬Agar (for plates)

• Adjust pH to 7.0

 

8.1 TAE Buffer:

For 1 L of 50 x TAE buffer you need:

• 242.48 g Tris

• 41.02 g Sodiumacetate

• 18.612 g EDTA

• Adjust pH to 7.8

• Solve in dH2O

20 mL of the stock is diluted in 1 L dH2O for the gel electrophoresis.

8.2 DNA Loading Buffer

• 50 % (v/v) glycerol

• 1 mM EDTA

• 0.1 % (w/v) bromphenol blue

• Solve in ddH20

Gel Preparation (%1 gel)

• Add 0,5 gr agarose to 50 ml TAE buffer.

• Until agorose being melted, heat it in microwave and do not forget to stir it often.

• After melting, it should be cool enough to handle it.

• Add 5 ml EtBr.

• Then, pour it to container and pay attention not to form bubbles in gel.

 

Gel Photo Imaging:

• Adjust zoom and position using visible light• Before turning on UV load your settings file which has the following parameters:

• Preview tab, all three options checked

• Active image

• Dynamic integration, auto exposure, 10 frames

• 50/50 brightness/contrast

• Maximize brightness with camera knob (counterclockwise)

• Turn on UV light

• Lower brightness from camera knob if necessary

Gel Extraction :

We used QIAGEN kit do with the following changes:

http://kirschner.med.harvard.edu/files/protocols/QIAGEN_QIAquickSpin_EN.pdf

Changes in that protocol:

At step 0: Wear disposable lab coat or long sleeves to prevent UV-burn. Use a razor

blade/scalpel to excise the band.

At step 4: Add 10 ul for 3M NaOAc no matter if the color is yellow or not. This will correct the

pH and should turn the QG back to yellow.

At step 7: Combine samples of identical DNA in the same column (spin multiple times if

necessary).

At step 13: Elute in 30 ul rather than 50 ul to really maximize yield.