Haynes:PCR clip clone

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Type IIS Assembly

by Karmella Haynes, 2013

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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• Clean up PCR products
  • Zymo clean and concentrator
  • Elute w/ 25 μL dH₂O

• Assemblies

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

• Digests
  
  • E/X

1. AubR, 851 bp
2. BjaR, 776 bp
3. BraR, 776 bp
4. RpaR, 779 bp
5. Modular Receiver Vector (MRV)

Reagent	Rxn 1-4	Rxn 5
DNA	10.0	5.0
10X buffer	3.0	3.0
EcoRI	1.0	1.0
XbaI	1.0	1.0
dH2O	15.0	20.0
	30.0	30.0

• Column clean-up
  • Zymo clean and concentrator

• 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)