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Revision as of 05:38, 27 October 2010

Protocols

CPEC Cloning

Materials

• Phusion™ High-Fidelity PCR Kit (FINNZYMES, Cat. No. F-553)
• Thermocycler

Preparation

5x Phusion HF Buffer    4 ul
10 mM dNTPs	        0.4 ul	
Vector		        50 ng/1kb	
Insert		        x ng*	
Phusion DNA Polymerase  0.2 ul
-------------------------------
H2O		        to 20 ul

*The amount of insert is determined so that the molar ratio for vector and insert is 1 to 2.

Procedures

98°C                  30sec
  10X
     98°C             10 sec
     Annealing**      30 sec
     72°C             x sec***
72°C                  5min
4°C                   hold

** Anneal at Tm + 3°C. The Tm should be calculated with the nearest-neighbor method.
***The extension time is usually calculated according to the shortest piece with 15 sec /kb if the cloning is not complicated. For example, if there is only one insert and is shorter than the vector, say, 600 bp, then I will use 15 sec for extension. Refer to the published paper for detailed information.

Transformation

1: Thaw 1 tube of competent cells on ice;

2: Add 1-50ng of plasmid into competent cells while stirring gently;

3: Keep the tube covered by ice for 30min;

4: Heat-shock the competent cells in water bath for 45s at 42C;

5: Put the tube on ice for 2 min;

6: Add 450uL of SOC medium and put it in a 37C shaker for 1 hour;

7: Dilute and spread an appropriate amount on an LB agar plate with the appropriate antibiotcs;

8: Place the plate upside-down in a 37C incubator for 16-18 hours.

Plasmid Extraction (Miniprep)

1:Pick a single colony from a freshly streaked selective plate and inoculate a culture of 1–5 ML LB MEDIUM CONTAINING THE APPROPRIATE SELECTIVE ANTIBIOTIC. INCUBATE FOR 12–16 H AT 37‹C WITH VIGOROUS SHAKING;

2: THE BACTERIAL CELLS BY CENTRIFUGATION AT > 8000 RPM (6800 X G) IN A CONVENTIONAL, TABLE-TOP MICROCENTRIFUGE FOR 3 MIN AT ROOM TEMPERATURE (15–25‹);

3: RESUSPEND PELLETED BACTERIA IN 250 UL OF RESUSPENSION BUFFER AND TRANSFER TO A MICROCENTRIFUGE TUBE;

4: ADD 250 UL OF LYSIS BUFFER AND THOROUGHLY MIX BY INVERSION. IMPORTANT: DO NOT ALLOW LYSIS TO CONTINUE FOR MORE THAN 5 MIN;

5: ADD 350 UL OF NEUTRALIZATION BUFFER AND THOROUGHLY MIX BY INVERSION;

6: CENTRIFUGE FOR 10 MIN AT 13,000 RPM (ALL CENTRIFUGATION MAY BE PERFORMED AT 13,000 RPM);

7: DECANT SUPERNATANTS FROM STEP 6 INTO A SPIN COLUMN;

8: CENTRIFUGE FOR 30-60S AND DISCARD THE FLOW THROUGH;

9: ADD 750 UL OF WASH BUFFER AND CENTRIFUGE FOR 30-60S;

10: DISCARD FLOW-THROUGH, CENTRIFUGE FOR AN ADDITIONAL MINUTE TO REMOVE RESIDUAL WASH BUFFER;

11: PLACE THE SPIN COLUMN IN A CLEAN MICROCENTRIFUGE TUBE, ADD 20-50UL OF ELUTION BUFFER OR DDH2O TO THE CENTER OF THE SPIN COLUMN, LET IT STAND FOR 1 MIN AND CENTRIFUGE FOR 1MIN.

12: A SECOND ELUTION STEP MAY BE PERFORMED TO INCREASE DNA YIELDS.

Restriction Digest

Reaction Mix: •100 μg/mL BSA •1X NEB2 buffer •1 μL BioBrick enzyme 1 •1 μL BioBrick enzyme 2

DNA samples should be .2-1ng, and deionized, sterile H2O should be used to bring the solution to H2O to 50 μL

1.Add restriction enzyme buffer. Add BSA. Add DNA. Add each enzyme. Also, the enzyme is in some percentage of glycerol which tends to stick to the sides of your tip. To ensure you add only 1 μL, just touch your tip to the surface of the liquid when pipetting. Incubate for 2 hours at 37°C. Incubate for 20 mins at 80°C to heat inactivate enzyme. This step is sufficient to inactivate even Pst I.

2.Incubate 4°C until you pull the reaction out of the thermal cycler. Protocol from Openwetware:

http://openwetware.org/wiki/Engineering_BioBrick_vectors_from_BioBrick_parts/Restriction_digest

PCR

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Materials

• Phusion™ High-Fidelity PCR Kit (FINNZYMES, Cat. No. F-553)
• Thermocycler

Preperation

Procedure

98°C           30sec 
 30X
    98°C       10 sec
    Annealing* 30 sec
    72°C       15 sec per 1 kb
72°C           5min
4°C            hold

* Anneal at Tm + 3°C. The Tm should be calculated with the nearest-neighbor method.

Ligation

1.Add appropriate amount of deionized H2O to sterile 0.6 mL tube

Add 1 μL ligation buffer to the tube.

Vortex buffer before pipetting to ensure that it is well-mixed.

Remember that the buffer contains ATP so repeated freeze, thaw cycles can degrade the ATP thereby decreasing the efficiency of ligation.

Add appropriate amount of insert to the tube.

Add appropriate amount of vector to the tube.

Add 0.5 μL ligase.

Vortex ligase before pipetting to ensure that it is well-mixed.

Also, the ligase, like most enzymes, is in some percentage of glycerol which tends to stick to the sides of your tip. To ensure you add only 0.5 μL, just touch your tip to the surface of the liquid when pipetting.

Let the 10 μL solution sit at 22.5°C for 30 mins Denature the ligase at 65°C for 10min

Dialyze for 20 minutes if electroporating

Use disks shiny side up

2.Store at -20°C