1.PCR:

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

2.CULTURES:

2.1 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

2.2 Glycerol Stock:

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

·         Store at -80C

2.3 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.

2.4 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.

3.GELS:

3.1 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.

3.2 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

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.

6.RESTRICTION DIGESTION:

NEB Buffer  5 µl
BSA              0.5 µl
Enzyme 1         0.5 µl
Enzyme 2         0.5 µl
Plasmid* à 1000ng/ml

To complete to 50 µl, add ddH2O.
1- Keep it in waterbath for 2.5 hours at 37 C0.
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.

Calculating Insert Amount:

8.MATERIALS AND CHEMICALS:

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

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

9.QIAGEN Kits

9.1 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 the cells at 10000g for 1 min.

·         Discard supernatant.

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

·         Spin down the cells at 10000g for 1 min.

·         Discard supernatant.

·         Resuspend the cells with 250 µL p1* buffer and vortex.

·         Add 250 µL P2 buffer and make sure the blue color is equally spread. Gently inverse the eppendorf 4-5 times. DON’T VORTEX!!!

·         Wait 5 min at room temperature.

·         Add 350 µL N3 buffer (cold +4C°) inverse gently and immediately.

·         Incubate on ice for 15 min.

·         Spin down the cells at 10000g for 15 min at +4C°.

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

·         Spin down the cells at 10000g for 1 min.

·         Remove the below part of the tube and discard supernatant.

·         Add 500 µL PB buffer.

·         Add 750 µL PE buffer and wait 1 min near the flame.

·         Spin down the cells at 10000g for 1 min.

·         Spin down the cells at 13000g for 1 min again.

·         Wait 5 min near the flame.

·         Put the filter parts onto the 1,5 ml eppendorfs.

·         Add 50 µL elution buffer.

·         Wait 1 min near the flame.

·         Spin down the cells at 13000g for 1 min.

·         Store your plasmids at+4°C for 1 hour before further experiments.

9.2 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.

9.3 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.

10.Competent Cells:

      Materials:

·         Detergent-free, sterile glassware and plasticware

·         Table-top OD600nm spectrophotometer

·         1 ml DH5 alpha cells

·         LB

·         0,1M CaCI2                                                                                                                                     

     Preparation:

·         Take 1ml from the DH5 alpha cells which were grown one day ago and put it in to 100ml LB medium. Keep it at 370C for 2 hours and measure it by spectrophotometer at 600 nm wavelength until absorbance reaches OD  of 0.375.

·         Divide 100 ml sample into 2 falcon and centrifuge for 10 min at 5000 rpm at +40C.

·         Discard the supernatant.

·         Add 10 ml 0.1 M cold CaCl2 into supernatant and dissolve the pellet. Put it on ice for 10 min.

·         Centrifuge at 5000 rpm for 5 min at +40C. Then, discard the supernatant.

·         Add 10 ml CaCl2 and dissolve it by shaking it up and down.

·         Put the sample on ice for half an hour.

·         Centrifuge at 5000 rpm for 5 min at +4°C .Then, discard the supernatant.

·         Put 2 ml CaCl2 and dissolve the pellet.

·         Put it on ice for 5 min and keep it at +40C.

11. 3A Assembly Kit:

We used the linear plasmids and the procedure that the iGEM provides us;

11.1 Digestion:

• Enzyme Master Mix for Plasmid Backbone (25ul total, for 6 rxns)
• 5 ul NEB Buffer 2
• 0.5 ul BSA
• 0.5 ul EcoRI-HF
• 0.5 ul PstI
• 0.5 ul DpnI (Used to digest any template DNA from production)
• 18 ul dH20
• Digest Plasmid Backbone
• Add 4 ul linearized plasmid backbone (25ng/ul for 100ng total)
• Add 4 ul of Enzyme Master Mix
• Digest 37C/30 min, heat kill 80C/20 min

11.2 Ligation:

• Add 2ul of digested plasmid backbone (25 ng)
• Add equimolar amount of EcoRI-HF SpeI digested fragment (< 3 ul)
• Add equimolar amount of XbaI PstI digested fragment (< 3 ul)
• Add 1 ul T4 DNA ligase buffer.
•  Note: Do not use quick ligase
• Add 0.5 ul T4 DNA ligase
• Add water to 10 ul
• Ligate 16C/30 min, heat kill 80C/20 min
• Transform with 1-2 ul of product

References:12.