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| This protocol describes how to carry out asymmetric PCR and single-sided PCR. Both types of PCR are used to produce single-stranded DNA. Asymmetric PCR initially generates a substantial amount of double-stranded DNA and the double-stranded DNA is then used to generate single-stranded DNA. This approach is especially useful for direct sequencing of PCR products. Single-sided PCR uses only a single primer to generate single-stranded fragments either for sequencing purposes or for use as single-stranded probes. Single-sided PCR is carried out in the same manner as ordinary PCR except that one of the primers is left out of the reaction. |
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A. Asymmetric PCR
1. Pick a phage plaque and place in 100 μl TE or scrape a fresh colony of a bacterial transformant of choice and place in 50 μl of TE/TX100 in a microcentrifuge tube.
2. Heat the tube for 10 min at 95°C.
3. Centrifuge at maximum speed for several minutes in a microcentrifuge to pellet cell debris. Collect the supernatant.
4. Add the following components in a PCR tube:
5 μl of phage or bacterial extract (from Step #A3)
50 μM of dNTPs
50 pmol of Primer 1
1 pmol of Primer 2
in 1X PCR Reaction Buffer to give a final reaction volume of 50 to 100 μl
2.5 Units of Taq polymerase
5. Run 30 to 35 cycles in a thermocycler using the following PCR program (see Hint #1 and #2)
95°C for 60 sec
60°C for 30 sec
72°C for 2 min
6. Run a small aliquot on an agarose gel to analyze for single-stranded DNA (see Protocol on Agarose Gel Electrophoresis of DNA).
7. Purify the PCR products and sequence, if desired.
B. Single-sided PCR
1. Pick a phage plaque and place in 100 μl of TE or scrape a fresh colony of a bacterial transformant of choice and place in 50 μl of TE/TX100 in a microcentrifuge tube.
2. Heat the tube for 10 min at 95°C.
3. Centrifuge at maximum speed for several minutes in a microcentrifuge to pellet cell debris. Collect the supernatant.
4. Add the following components in a PCR tube:
5 μl of phage or bacterial extract (from Step #B3)
50 μM dNTPs
50 pmol Primer 1
make in 1X PCR Reaction Buffer to give a final reaction volume of 50 to 100 μl
2.5 Units of Taq Polymerase
5. Run 30-35 cycles in a thermocycler using the following PCR program (see Hint #1 and #2)
95°C for 60 sec
60°C for 30 sec
72°C for 2 min
6. Run a small aliquot on an agarose gel to analyze for single-stranded DNA (see Protocol on Agarose Gel Electrophoresis of DNA).
7. Purify the PCR products and sequence, if desired.
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| 500 μM dNTPs |
| 500 μM TTP
500 μM ATP
500 μM GTP
500 μM CTP
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| PCR Reaction Buffer (10X) |
| 15 mM MgCl2
500 mM KCl
200 mM Tris-Cl, pH 8.5
1% (v/v) Tween 20
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| TE/TX100 |
| 1% (v/v) Triton-X 100
20 mM Tris-Cl, pH 8.5
2 mM EDTA
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| TE |
| 1 mM EDTA
10 mM Tris-Cl, pH 8.5
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GTP
CTP
TTP
Tris
Oligonucleotide
DNA Polymerase, Taq
ATP
Primer
EDTA
Magnesium Chloride
Tween 20
Triton X-100
Potassium Chloride
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1. Use 35 cycles for the phage extract.
2. The annealing temperature may need to be adjusted based on the melting temperature (Tm) of your primers.
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