Overlap‐elongation rolling circle amplification

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.