Haynes:PCR clip clone

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PCR, Clip, and Clone

by Karmella Haynes, 2015

Principle: The insert is PCR-amplified using an error-reducing, high-fidelity polymerase (Phusion) and primers that add restriction sites to each end of the insert. The PCR product is cleaned up using a DNA-binding column. A predetermined amount of clean DNA is cut at the ends (or "clipped") and the 5'phosphates are removed in a 20 μL-reaction. Enzymes are deactivated at 75°C and a small amount of the reaction is used directly as insert in a ligation.

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Plan the DNA Ligations
In your notebook, make a list of all insert+vector and vector-only controls that you will set up ligations for. Include...

1. a short name for the construct
2. a short name for the insert, what it will be cut with (in parentheses), and its length in bp
3. a plus sign followed by the vector, what it will be cut with (in parentheses), and its length in bp

EXAMPLE List: In line 1, the short name for the construct is AubR/MRV. The insert is Aub-PCR cut with EcoRI and XbaI, and it is 851 bp long. The vector is MRV, cut with EcoRI and XbaI, and it is 3201 bp long. The last line is the negative control. There is only one negative control because only one vector is being used for all of the ligations.

1. AubR/MRV: AubR-PCR(E/X)/851 + MRV(E/X)/3201
2. BjaR/MRV: BjaR-PCR(E/X)/776 + MRV(E/X)/3201
3. BraR/MRV: BraR-PCR(E/X)/776 + MRV(E/X)/3201
4. RpaR/MRV: RpaR-PCR(E/X)/779 + MRV(E/X)/3201
5. Neg ctrl: MRV(E/X)/3201

Phusion PCR

1. Design insert amplification primers that have the appropriate restriction sites on the ends. The restriction sites must be ABSENT from the insert sequence itself.
2. Set up a Phusion PCR reaction as shown in the table below. Use the table as a template to record your work in your notebook.
   1. Note: Use 50 - 100 ng of template DNA is the template is a plasmid. Use more DNA if the specific target is at a lower copy number (e.g. GAPDH gene in genomic DNA, ACTB gene in cDNA library, etc.).

Reagent	Volume	Expected: 1. Insert 1 = ### bp 2. Insert 2 = ### bp 3. etc.	Example Image from 04/17/15; 5 μL/lane; 1% agarose; Ladder
DNA template*	0.5 μL
10 μM F primer	1.0
10 μM R primer	1.0
10 mM dNTPs	1.0
Phusion Pol.	0.5
5X HF buffer	10.0
dH2O	36.0
	50.0

Program:

• 98°C, 3 min
• 35x[98°C, 10 sec; 66°C, 30 sec; 72°C, 60 sec]
• 72°C, 10 min
• 4°C ∞

Clean up PCR product(s)

1. Use the Qiagen PCR Clean-up kit to purify the amplified DNA.
2. In the final elution step, use 30 μL of elution solution.
3. Measure the concentration(s) of the sample(s).
4. Use the table below as a template to record your results in your notebook.

Sample	OD260	260/280	ng/μL
1. Insert 1 PCR	###	###	###
2. Insert 2 PCR	###	###	###
3. etc.	###	###	###

Digest the Insert(s)

1. Set up restriction digests as shown in the table below

Reagent	Volume
DNA (500 ng)	up to 15.0 μL
10X buffer	3.0
EcoRI	1.0
XbaI	1.0
dH2O	x μL
	20.0

• Measure conc.'s

Sample	OD260	260/280	ng/μL
1. AubR-PCR (E/X)	-0.005	5.875	-5.001
2. BjaR-PCR (E/X)	-0.005	9	-4.788
3. BraR-PCR (E/X)	-0.005	7.143	-5.291
4. RpaR-PCR (E/X)	-0.003	29	-3.052
5. MRV (E/X)	0.007	1.388	7.11

• Note: unsure why concentrations so low. Proceed with ligations anyway, see what happens.

• Ligations

	Rxn 1-4	Neg
Insert DNA	3.0	---
Vector DNA (25 ng)	3.5	3.5
2x lgn buf (Roche)	7.5	7.5
T4 ligase (NEB)	1.0	1.0
dH2O	---	3.0
	15 μL	15 μL

• Transform 50 uL DH5α-turbo
• Plate on 100 μg/mL amp

RESULTS (4/22/15)

1. AubR/MRV: 11 colonies
2. BjaR/MRV: 11 colonies
3. BraR/MRV: 6 colonies
4. RpaR/MRV: 7 colonies
5. MRV (neg): 3 colonies

• Success! Pick 2 colonies from plates 1-4 for streak plate and liquid cultures (5 mL each)