1.PCR:

1.1.Colony PCR:

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

(For 1x sample preparation)

׼span style="font:7.0pt "Times New Roman"">         dNTP              1 ֌

׼span style="font:7.0pt "Times New Roman"">         Buffer             5 ֌

׼span style="font:7.0pt "Times New Roman"">         MgCl₂             3 ֌

׼span style="font:7.0pt "Times New Roman"">         **Forward Primer       0,5 ֌

׼span style="font:7.0pt "Times New Roman"">         **Reverse Primer        0,5 ֌

׼span style="font:7.0pt "Times New Roman"">         Template         -

׼span style="font:7.0pt "Times New Roman"">         Taq Polymerase                       0,2 ֌

׼span style="font:7.0pt "Times New Roman"">         dH₂O              39,8 ֌

׼span style="font:7.0pt "Times New Roman"">         TOTAL           50 ֌

* 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:

׼span style="font:7.0pt "Times New Roman"">         Use 1ul of 60ng/ul or 100ng/ul DNA

׼span style="font:7.0pt "Times New Roman"">         Use 1ul of each primer at 3.2pmole/ul concentration or 1.25ul of each primer at 100ng/ul concentration

׼span style="font:7.0pt "Times New Roman"">         2.5ul 10x PCR Buffer w/ Mg (1.5mM)

׼span style="font:7.0pt "Times New Roman"">         0.5ul 25mM MgCl2

׼span style="font:7.0pt "Times New Roman"">         0.5ul dNTP

׼span style="font:7.0pt "Times New Roman"">         0.125ul Taq

׼span style="font:7.0pt "Times New Roman"">         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:

׼span style="font:7.0pt "Times New Roman"">         95м/span>C for 1-5minutes (usually 4min)

׼span style="font:7.0pt "Times New Roman"">         95м/span>C for 1min

׼span style="font:7.0pt "Times New Roman"">         55м/span>C for 1min                                              Cycle 30 times

׼span style="font:7.0pt "Times New Roman"">         72м/span>C for 1.5 to 2min (usually 2min)

׼span style="font:7.0pt "Times New Roman"">         72м/span>C for 10min

׼span style="font:7.0pt "Times New Roman"">         4м/span>C hold

2.CULTURES:

2.1 Liquid Culture:

׼span style="font:7.0pt "Times New Roman"">         5 ml LB is mixed with appropriate antibiotic and a culture tube is prepared with certain labels.

׼span style="font:7.0pt "Times New Roman"">         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

׼span style="font:7.0pt "Times New Roman"">         Incubate the culture at 37у for 13-15 hours. Do not let the cells become old to not release their plasmids

׼span style="font:7.0pt "Times New Roman"">         If the culture becomes saturated: you can reinoculate 30֌ into a new 3mL LB tube

2.2 Glycerol Stock:

׼span style="font:7.0pt "Times New Roman"">         Put 500֌ of a mid-log culture into a 1.5mL tube with 500֌ 80% glycerol

׼span style="font:7.0pt "Times New Roman"">         Store at -80C

2.3 Spreading Plates:

׼span style="font:7.0pt "Times New Roman"">         Put 50 ֌ and 150 ֌ of whether LB culture or transformed cells.

׼span style="font:7.0pt "Times New Roman"">         Spread the cells into the plate until there is no seen liquid.

׼span style="font:7.0pt "Times New Roman"">         Wait until the plates dried.

׼span style="font:7.0pt "Times New Roman"">         After 14-16 hours colonies will show up.

2.4 Streaking Plates:

׼span style="font:7.0pt "Times New Roman"">         Put a drop your culture on the plate

׼span style="font:7.0pt "Times New Roman"">         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)

׼span style="font:7.0pt "Times New Roman"">         Wait until the plates dried.

׼span style="font:7.0pt "Times New Roman"">         After 14-16 hours colonies will show up.

3.GELS:

3.1 Gel Preparation (%1 gel)

׼span style="font:7.0pt "Times New Roman"">         Add 0,5 gr agarose to 50 ml TAE buffer.

׼span style="font:7.0pt "Times New Roman"">         Until agorose being melted, heat it in microwave and do not forget to stir it often.

׼span style="font:7.0pt "Times New Roman"">         After melting, it should be cool enough to handle it.

׼span style="font:7.0pt "Times New Roman"">         Add 5 ml EtBr.

׼span style="font:7.0pt "Times New Roman"">         Then, pour it to container and pay attention not to form bubbles in gel.

3.2 Gel Photo Imaging:

׼span style="font:7.0pt "Times New Roman"">         Adjust zoom and position using visible light

׼span style="font:7.0pt "Times New Roman"">         Before turning on UV load your settings file which has the following parameters:

׼span style="font:7.0pt "Times New Roman"">         Preview tab, all three options checked

׼span style="font:7.0pt "Times New Roman"">         Active image

׼span style="font:7.0pt "Times New Roman"">         Dynamic integration, auto exposure, 10 frames

׼span style="font:7.0pt "Times New Roman"">         50/50 brightness/contrast

׼span style="font:7.0pt "Times New Roman"">         Maximize brightness with camera knob (counterclockwise)

׼span style="font:7.0pt "Times New Roman"">         Turn on UV light

׼span style="font:7.0pt "Times New Roman"">         Lower brightness from camera knob if necessary

4.GETTING THE DNA PARTS FROM KIT PLATE:

׼span style="font:7.0pt "Times New Roman"">         10 ul ddHO is added into the kit plate target part.

׼span style="font:7.0pt "Times New Roman"">         Wait 5 min.and get the part by well pipetting.

5.TRANSFORMATION:

      Materials:

׼span style="font:7.0pt "Times New Roman"">         Resuspended DNA (2 ul )

׼span style="font:7.0pt "Times New Roman"">         Competent cells (50ul per transformation)

׼span style="font:7.0pt "Times New Roman"">         Ice

׼span style="font:7.0pt "Times New Roman"">         2ml tube (1 per a transformation')

׼span style="font:7.0pt "Times New Roman"">         42ۃ water bath

׼span style="font:7.0pt "Times New Roman"">         Petri dishes with LB agar and appropriate antibiotic (2 or 3 per transformation)

׼span style="font:7.0pt "Times New Roman"">         Glass beads or spreader

׼span style="font:7.0pt "Times New Roman"">         37ۃ incubator

      Preparation:

׼span style="font:7.0pt "Times New Roman"">         Start thawing the competent cells on ice.

׼span style="font:7.0pt "Times New Roman"">         Add 50 ֌ of thawed competent cells into pre-chilled 2ml tube.

׼span style="font:7.0pt "Times New Roman"">         Add 1 - 2 ֌ 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.

׼span style="font:7.0pt "Times New Roman"">         Close tube and incubate the cells on ice for 30 minutes.

׼span style="font:7.0pt "Times New Roman"">         Heat shock the cells by immersion in a pre-heated water bath at 42ۃ for 60 seconds.

׼span style="font:7.0pt "Times New Roman"">         Incubate the cells on ice for 5 minutes.

׼span style="font:7.0pt "Times New Roman"">         Add 400 ֬ of LB media (make sure that the broth does not contain antibiotics and is not contaminated)

׼span style="font:7.0pt "Times New Roman"">         Incubate the cells at 37ۃ 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.

׼span style="font:7.0pt "Times New Roman"">         Label two petri dishes with LB agar and the appropriate antibiotic(s) with the part number, plasmid backbone, and antibiotic resistance. Plate 20 ֬ and 200 ֬ of the transformation onto the dishes, and spread. This helps ensure that you will be able to pick out a single colony.

׼span style="font:7.0pt "Times New Roman"">         Incubate the plate at 37ۃ 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.

׼span style="font:7.0pt "Times New Roman"">         You can pick a single colony, make a glycerol stock, grow up a cell culture.

6.RESTRICTION DIGESTION:

NEB Buffer  5 ֬
BSA              0.5 ֬
Enzyme 1         0.5 ֬
Enzyme 2         0.5 ֬
Plasmid* ༯span> 1000ng/ml

To complete to 50 ֬, add ddH2O.
1- Keep it in waterbath for 2.5 hours at 37у.
2- Run the samples on the gel. 

7.LIGATION:

׼span style="font:7.0pt "Times New Roman"">         10X T4 ligase buffer: 2.0 ֌

׼span style="font:7.0pt "Times New Roman"">         6:1 molar ratio of insert to vector (~10ng vector)

׼span style="font:7.0pt "Times New Roman"">         Add (8.5 - vector and insert volume)֬ ddH2O

׼span style="font:7.0pt "Times New Roman"">         T4 Ligase: 1 ֌

׼span style="font:7.0pt "Times New Roman"">         Incubation at room temperature 1 hour and 15 min at 65у for enzyme inactivation.

׼span style="font:7.0pt "Times New Roman"">         Alternatively: Incubation at +4у 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.

׼span style="font:7.0pt "Times New Roman"">         Take 2 ml cell in to the eppendorf (2ml).

׼span style="font:7.0pt "Times New Roman"">         Spin down the cells at 10000g for 1 min.

׼span style="font:7.0pt "Times New Roman"">         Discard supernatant.

׼span style="font:7.0pt "Times New Roman"">         Add 2 ml cell in to the eppendorf again, totally 4 ml will be pelleted.

׼span style="font:7.0pt "Times New Roman"">         Spin down the cells at 10000g for 1 min.

׼span style="font:7.0pt "Times New Roman"">         Discard supernatant.

׼span style="font:7.0pt "Times New Roman"">         Resuspend the cells with 250 ֌ p1* buffer and vortex.

׼span style="font:7.0pt "Times New Roman"">         Add 250 ֌ P2 buffer and make sure the blue color is equally spread. Gently inverse the eppendorf 4-5 times. DONӔ VORTEX!!!

׼span style="font:7.0pt "Times New Roman"">         Wait 5 min at room temperature.

׼span style="font:7.0pt "Times New Roman"">         Add 350 ֌ N3 buffer (cold +4у) inverse gently and immediately.

׼span style="font:7.0pt "Times New Roman"">         Incubate on ice for 15 min.

׼span style="font:7.0pt "Times New Roman"">         Spin down the cells at 10000g for 15 min at +4у.

׼span style="font:7.0pt "Times New Roman"">         Place the supernatant into the tube that is placed into the kit.

׼span style="font:7.0pt "Times New Roman"">         Spin down the cells at 10000g for 1 min.

׼span style="font:7.0pt "Times New Roman"">         Remove the below part of the tube and discard supernatant.

׼span style="font:7.0pt "Times New Roman"">         Add 500 ֌ PB buffer.

׼span style="font:7.0pt "Times New Roman"">         Add 750 ֌ PE buffer and wait 1 min near the flame.

׼span style="font:7.0pt "Times New Roman"">         Spin down the cells at 10000g for 1 min.

׼span style="font:7.0pt "Times New Roman"">         Spin down the cells at 13000g for 1 min again.

׼span style="font:7.0pt "Times New Roman"">         Wait 5 min near the flame.

׼span style="font:7.0pt "Times New Roman"">         Put the filter parts onto the 1,5 ml eppendorfs.

׼span style="font:7.0pt "Times New Roman"">         Add 50 ֌ elution buffer.

׼span style="font:7.0pt "Times New Roman"">         Wait 1 min near the flame.

׼span style="font:7.0pt "Times New Roman"">         Spin down the cells at 13000g for 1 min.

׼span style="font:7.0pt "Times New Roman"">         Store your plasmids at+4у 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.

׼span style="font:7.0pt "Times New Roman"">         3 volumes of Buffer QG is added to 1 volume of the PCR sample and mixed by vortexing.

׼span style="font:7.0pt "Times New Roman"">         1 volume of the INITIAL sample volume of isopropanol is added to the sample and mix.

׼span style="font:7.0pt "Times New Roman"">         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 30ֶ0 s.Discard flow-through. Place the QIAquick column back into the same tube

׼span style="font:7.0pt "Times New Roman"">         Add 0.75 ml Buffer PE to the QIAquick column with the pipette and centrifuge for 30ֶ0 s. Discard flow-through and place the QIAquick column back in the same tube.

׼span style="font:7.0pt "Times New Roman"">         Centrifuge the column for an additional 1 min.

׼span style="font:7.0pt "Times New Roman"">         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).

׼span style="font:7.0pt "Times New Roman"">         To elute DNA, add 50 ֬ Buffer EB (10 mM Tris؃l, 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:

׼span style="font:7.0pt "Times New Roman"">         Detergent-free, sterile glassware and plasticware

׼span style="font:7.0pt "Times New Roman"">         Table-top OD600nm spectrophotometer

׼span style="font:7.0pt "Times New Roman"">         1 ml DH5 alpha cells

׼span style="font:7.0pt "Times New Roman"">         LB

׼span style="font:7.0pt "Times New Roman"">         0,1M CaCI₂                                                                                                                                     

     Preparation:

׼span style="font:7.0pt "Times New Roman"">         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.

׼span style="font:7.0pt "Times New Roman"">         Divide 100 ml sample into 2 falcon and centrifuge for 10 min at 5000 rpm at +4у.

׼span style="font:7.0pt "Times New Roman"">         Discard the supernatant.

׼span style="font:7.0pt "Times New Roman"">         Add 10 ml 0.1 M cold CaCl₂ into supernatant and dissolve the pellet. Put it on ice for 10 min.

׼span style="font:7.0pt "Times New Roman"">         Centrifuge at 5000 rpm for 5 min at +40C. Then, discard the supernatant.

׼span style="font:7.0pt "Times New Roman"">         Add 10 ml CaCl2 and dissolve it by shaking it up and down.

׼span style="font:7.0pt "Times New Roman"">         Put the sample on ice for half an hour.

׼span style="font:7.0pt "Times New Roman"">         Centrifuge at 5000 rpm for 5 min at +4у .Then, discard the supernatant.

׼span style="font:7.0pt "Times New Roman"">         Put 2 ml CaCl₂ and dissolve the pellet.

׼span style="font:7.0pt "Times New Roman"">         Put it on ice for 5 min and keep it at +4у.

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 16у/30 min, heat kill 80у/20 min
• Transform with 1-2 ul of product

References:12.