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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

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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.

DNA Purification Materials :  E.Z.N.A Gel Purification Kit (Omega Bio-Tek, Cat No. D2500-02 )  Water bath equilibrated to 55-65C  Microcentrifuge capable of at least 10,000 x g  Nuclease-free 1.5 ml centrifuge bottles  Absolute (95%-100%) ethanol  Protective eye-wear  Isopropanol (for fragments < 500 bp only) Protocol: 1. Perform agarose gel electrophoresis to fractionate DNA fragments. Any type or grade of agarose may be used. It is strongly recommended, however, that fresh TAE buffer or TBE buffer be used as running buffer. Do not re-use running buffer as its pH will increase and reduce yields. 2. When adequate separation of bands has occurred, carefully excise the DNA fragment of interest using a wide, clean scalpel. 3. Determine the approximate volume of the gel slice by weighing it in a clean 1.5 ml microfuge tube. Assuming a density of 1 g/ml of gel, the volume of gel is derived as follows: A gel slice of mass 0.3 g will have a volume of 0.3 ml. Add equal volume of Binding Buffer (XP2). Incubate the mixture at 55C-60C for 7 min or until the gel has completely melted. Mix by shaking or inverting the tube every 2-3 minutes. Centrifuge the tube briefly to collect all the liquid to the bottom of the tube. Note: For DNA fragment less than 500bp, add 1 sample volume of isopropanol after the addition of Binding Buffer (XP2). 1. Apply up to 700 ul of the DNA/agarose solution to a HiBind® DNA spin column assembled in a clean 2 ml collection tube (provided) and centrifuge in a microcentrifuge at 8,000-10,000 x g for 1 min at room temperature. Discard the liquid. Re-use the collection tube in Steps 5-8. For volumes greater than 700 ul, load the column and centrifuge successively, 700 ul at a time. Each HiBind® spin-column has a total capacity of 25-30 ug DNA. 2. Discard liquid and add 300ul Binding Buffer. Centrifuge at 10,000 x g for 1 minutes. 3. Add 700 ul of SPW Buffer diluted with absolute ethanol into the column and wait 2-3 minutes. Centrifuge at 10,000 x g for 1 min at room temperature to wash the sample. 4. Discard liquid and repeat step 6 with another 700 ul SPW Buffer. 5. Discard liquid and, re-using the collection tube, centrifuge the empty column for 1 min at maxi speed (>13,000 x g) to dry the column matrix. This drying step is critical for good DNA yields. 6. Place column into a clean 1.5 ml microcentrifuge tube (not provided). Add 30-50 ul depending on desired concentration of final product) Elution Buffer (or sterile deionized water) directly to the center of the column matrix, then incubate for 1 minute. Centrifuge 1 min at maxi speed (>13,000 x g) to elute DNA. This represents approximately 70% of bound DNA. PCR Materials  Phusion™ High-Fidelity PCR Kit (FINNZYMES, Cat. No. F-553)  Thermocycler Preperation 5x Phusion HF Buffer 10 ul 10 mM dNTPs 1 ul DNA template 1 pg – 10 ng Forward primer (10 uM) 2.5 ul Reverse primer (10 uM) 2.5 ul Phusion DNA Polymerase 0.5 ul

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H2O to 50 ul

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

Transformation: Materials:  TOP10 Chemical Competent Cells (Invitrogen, Cat No. C4040-03)  SOC Medium (Sigma, Cat. No. S1797)  LB Agar (Sigma, Cat. No. L3027)  Petri Dishes (VWR, Cat. No. SC25373-187)  Cell Spreader (VWR, Cat. No. 89042-018)  37°C incubator  37°C shaker  water bath Protocol: 1. Thaw 1 tube of competent cells on ice; 2. Add 3 ul of cloning product or 1-50 ng 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 45 sec at 42°C; 5. Put the tube on ice for 2 minutes; 6. Add 450 ul of SOC medium and then put it in a 37°C shaker for 1 hour; 7. Dilute and spread an appropriate amount on an LB agar plate with the appropriate antibiotics; 8. Place the plate up-side-down in 37°C incubator for 16-18 hours (overnight).