Editing Overlap‐elongation rolling circle amplification (section)

= Overlap‐elongation rolling circle amplification (OERCA)<br><br>  =

== Circulation of single stranded DNA<br><br>  ==

1. Add 250 pmol of oligo (25 ul of 10 uM) to&nbsp; a PCR tube.<br> 

2. Add 5 μL of 10X buffer.<br> 

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

5. Purify with spin column.<br> 

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

7. Purify again.<br> 

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

== OERCA<br>  ==

'''1. ''''''OERCA'''<br>

Add 150 ng of circular ssDNA template,<br> 

10‐40 pmol&nbsp; of sense and antisense DNA primers<br> 

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

5 ul 10X Buffer<br>water to 50 μL<br> 

Cycles:<br> 

Incubate at 95 °C for 2 min<br> 

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

72 °C for 5 min.<br> 

Purify on&nbsp; 1% agarose gel. Cut out ~400 to 1500 bp DNA (smear)<br> 

<br> 

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

<br>

'''2. Optional: for larger fragments.'''<br> 

Use above purified DNA<br> 

Incubate at 95 °C for 2 min<br> 

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

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

5. Ligation<br> 

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

5 units of T4 DNA ligase,<br> 

2 μl PEG‐4000 or PEG-6000, <br> 

2X ligation buffer, <br> 

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

ligate for 3 h and then transform NEB Turbo.<br><br><br> 

== PCR Screening&nbsp; of colonies<br>  ==

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. 

<br>Run on 1% agarose gel<br> 

Positive clones (inserts in the right orientation):&nbsp; large smears on gel.<br><br> 

== Clone verification by Digestion<br>  ==

Use BamH1 + EcoR1 = insert size<br> 

Sequencing positive clones 

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<br> 

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== OE-PCR ==

&nbsp;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<br>10X Phusion buffer, and water for a final volume of 40 μl.



PCR 

95 °C for a 2 min&nbsp;

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



Purify PCR product on&nbsp; 1% agarose gel.

<br>to get large fragments,&nbsp; do additional PCR cycles (up to 30 additional PCR cycles.<br><br>

[[Category:Protocols]]