Cloning Protocol

Molecular cloning is similar to polymerase chain reaction (PCR) in that it permits the replication of DNA sequence. The fundamental difference between the two methods is that molecular cloning involves replication of the DNA in a living microorganism, while PCR replicates DNA in an in vitro solution, free of living cells.

Step 1: Phusion PCR

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Do a 200 uL reaction for the insert (2 PCR tubes with 100 uL)

PCR Mix

Liquids	Amounts
10 uM forward primer	10 uL
10 uM reverse primer	10 uL
2x phusion master mix (MM)	50 uL
10 ng total template	2 uL total
H2O up to 200 uL	variable - top to 100 uL

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To calculate annealing temperature, use T_m, lower + 3⁰ C

PCR CYCLE

STEP	TEMP	TIME
Initial Denaturation	98⁰ C	30 s
25-35 cycles	98⁰ C	5-10 s
	45-72⁰ C annealing	10-30 s
	72⁰ C extension	15-30 s/kb
Final extension	72⁰ C	5-10 min
Hold	4⁰ C	inf

Step 2: Digestion of vector

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Digest 10 ug of vector to linearize while your PCR is ongoing. You can gel purify though if you're using 2 different restriction enzymes this is not required.

Try for 30 uL total.

5 ug vector

Amounts	Liquids
1 uL	Sph1
1 uL	Xba1
3 uL	Cutsmart
2 uL	H20
22 uL	DNA
30 uL	TOTAL

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