Paul.xu at 20:54, 17 July 2013

2013-07-17T20:54:20Z

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= Overlap‐elongation rolling circle amplification (OERCA) =

== Circulation of single stranded DNA ==

1. Add 250 pmol of oligo (25 ul of 10 uM) to  a PCR tube. 

2. Add 5 μL of 10X buffer. 

3. Add 2.5 μL Circligase enzyme mix, 2.5 μL MnCl2 and 2.5 μL ATP. 4. Incubated at 60 °C for 2 h. 

5. Purify with spin column. 

6. Incubate with 20 units of Exonuclease I and 1X Exonuclease buffer at 37 °C for 1 h. 

7. Purify again. 

8. Optional run an aliquote on 2% agarose to check purity. 

== OERCA ==

'''1. ''''''OERCA''' 

Add 150 ng of circular ssDNA template, 

10‐40 pmol  of sense and antisense DNA primers 

2.5 mM of a 70% G/C dNTP mixture 1μl of Phusion DNA polymerase 

5 ul 10X Buffer water to 50 μL 

Cycles: 

Incubate at 95 °C for 2 min 

30‐40 cycles at 95 °C for 20 s, 55°C for 20 s and 72 °C for 30 s 

72 °C for 5 min. 

Purify on  1% agarose gel. Cut out ~400 to 1500 bp DNA (smear) 

 

The PCR reaction conditions were 95 °C for 2 min for initial

denaturation, followed by 30-40 cycles at 95 °C for 20 s, 52-55°C for 20 s and 72 °C for 30 s, followed by a final 5 min extension at 72 °C. The resulting PCR product was purified using a PCR purification kit and visualized on a 1% agarose gel stained with SYBR® Safe DNA stain. For the synthesis of repetitive genes encoding for aELPs, we typically performed one initial round of OERCA as indicated above, followed by an OE-PCR-only step, in which OERCA products were further extended in the absence of primers at an annealing temperature of 60 °C. 

 

'''2. Optional: for larger fragments.''' 

Use above purified DNA 

Incubate at 95 °C for 2 min 

10‐15 cycles of 95 °C for 20 s,  60 °C, 72 °C for 5 min. 3. Digest 2 μg of 1783 pT7A31 with 2 μl of EcoRv for 2 hours, gel purify. 

4. Dephosphorylate EcoRv digested 1783 pT7A31 with  1 μl CIP calf intestinal alkaline phosphatase for 15 min to 1 h at 37°C, gel purify 

5. Ligation 

OERCA product ~210 ng, ~250 ng of digested vector,

5 units of T4 DNA ligase, 

2 μl PEG‐4000 or PEG-6000, 

2X ligation buffer, 

nuclease‐free water in a total volume of 20 μl.

ligate for 3 h and then transform NEB Turbo. 

== PCR Screening  of colonies ==

12.5 μl Taq green master mix,

10 pmol T7 primer,

10 pmol insert‐specific forward primer,

nuclease‐free water in a total volume of 25 μl.

PCR:

95 °C for 2 min for initial denaturation

30 cycles at 95 °C for 30 s, 52 °C for 30 s, and 72 °C for 1 min.

 Run on 1% agarose gel 

Positive clones (inserts in the right orientation):  large smears on gel. 

== Clone verification by Digestion ==

Use BamH1 + EcoR1 = insert size 

Sequencing positive clones

 

 

 

== OE-PCR ==

 The OE-PCR reaction mixture consisting of 10-40 pmolof each ssDNA oligonucleotide, 10 nmol of a dNTP mixture, 0.8 μl of Phusion polymerase, 4 μl of 10X Phusion buffer, and water for a final volume of 40 μl.

PCR

95 °C for a 2 min 

30 cycles at 95 °C for 20 s, 55°C for 20 s and 72 °C for 30 s, Final 5 min extension at 72 °C.

Purify PCR product on  1% agarose gel.

 to get large fragments,  do additional PCR cycles (up to 30 additional PCR cycles. 

[[Category:Protocols]]

Overlap‐elongation rolling circle amplification - Revision history

Paul.xu at 20:54, 17 July 2013