Jun 28, 2023
Version 2
Converting ssDNA oligos to dsDNA with T4 DNA polymerase V.2
- 1University of Toronto
Protocol Citation: Alex N Nguyen Ba 2023. Converting ssDNA oligos to dsDNA with T4 DNA polymerase. protocols.io https://dx.doi.org/10.17504/protocols.io.261ged6xyv47/v2Version created by Alex N Nguyen Ba
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 28, 2023
Last Modified: April 18, 2024
Protocol Integer ID: 84169
Abstract
This protocol allows one to convert ssDNA to dsDNA oligos. In principle, one can buy two complementary oligos and anneal them. However, there are a few cases where randomized bases are desired, and therefore complementary oligos can not be ordered. The protocol works similarly to PCR, except that the reaction is not performed at the high temperature of typical thermocycling, because the oligos are short and their melting temperature can be in the order of the extension temperature of high-fidelity polymerases. This protocol is therefore particularly useful when creating barcodes or when short dsDNA fragments containing degenerate bases are desired.
Protocol materials
NEBuffer 2.1 - 5.0 mlNew England BiolabsCatalog #B7202S
Step 5
dNTPs
Step 7
T4 DNA Polymerase - 750 unitsNew England BiolabsCatalog #M0203L
Step 9
EDTA (0.5 M), pH 8.0Life TechnologiesCatalog #AM9260G
Step 11
Monarch PCR and DNA Cleanup Kit - 250 prepsNew England BiolabsCatalog #T1030L
Step 12
Primer design
Primer design
Design your target single-stranded DNA oligo such that the 3' end contains the reverse complement of an extending oligonucleotide. For example, appending "GTCATAGCTGTTTCCTG" to the end of your oligo will allow an oligonucleotide matching the M13 Reverse (-27) primer to extend it (5'-CAGGAAACAGCTATGAC-3').
Resuspend your oligonucleotides in 1/10th TE (10mM Tris, 0.1mM EDTA pH 8) to a final concentration of 100 micromolar (µM) to form your stock oligonucleotide solutions.
Make working oligonucleotide solutions by diluting the stock oligonucleotides to a final concentration of 10 micromolar (µM) in water.
Oligo annealing
Oligo annealing
Mix in a PCR tube 5 µL of a 10 micromolar (µM) stock of the target oligo with 10 µL of a 10 micromolar (µM) stock of the extending oligo. Add 27ul of molecular biology grade water.
Add 5 µL of NEBuffer 2.1 - 5.0 mlNew England BiolabsCatalog #B7202S . Mix well by vortexing.
Place the tube in a thermocycler and run the following protocol:
1. 95 degrees for 10 seconds
2. Decrease by 1 degrees
3. Repeat step 2 every 10 seconds, for 90 cycles.
4. Hold at 4 degrees.
15m
Oligo extension
Oligo extension
Add 2.5 µL dNTPsContributed by users (10 millimolar (mM) stock) and mix well by vortexing.
Go to the thermocycler and prepare the reaction cycle. When ready, start the protocol and pause when the block reaches 0 degrees (when Step 1 begins).
1. 5 minutes at 0 degrees C.
2. 5 minutes at 22 degrees C.
3. 30 minutes at 37 degrees C.
4. Hold at 0 degrees.
40m
Add 0.5 µL of T4 DNA Polymerase - 750 unitsNew England BiolabsCatalog #M0203L and mix well.
Place the tube in the thermocycler and unpause the run.
Oligonucleotide cleanup
Oligonucleotide cleanup
10m
10m
As soon as the reaction is completed, add EDTA (0.5 M), pH 8.0Life TechnologiesCatalog #AM9260G to 10 millimolar (mM) .
Use a PCR purification kit to purify the oligo. Ensure the kit is able to purify short oligonucleotides if needed (a kit such as Monarch PCR and DNA Cleanup Kit - 250 prepsNew England BiolabsCatalog #T1030L can be suitable).
10m