Oct 13, 2024
PCR Fragment Generation (for inserts)
- 1Washington University
Protocol Citation: Carolina Lopez 2024. PCR Fragment Generation (for inserts). protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvw1dwzlmk/v1
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: May 28, 2024
Last Modified: October 13, 2024
Protocol Integer ID: 100762
Abstract
Protocol to generate inserts by PCR for cloning
Materials
Reagents:
- Taq Polymerase (Thermofisher, 11304011)
- dNTPs
- PCR Primers
- Plasmid
- Nuclease Free H20
- Wizard SV Gel and PCR Purification Kit (Promega, A9282)
- DpnI (NEB, R0176L)
- 10x Cutsmart Buffer
PCR Product (Insert generation)
PCR Product (Insert generation)
47m
47m
1. Set up PCR Reaction:
2. Run PCR Conditions:
a) 95 °C , 00:02:00
b) 95 °C , 00:00:30
c) 60 °C , 00:00:30
d) 72 °C , 00:02:00
e) Repeat step B-D 35 times
f) 72 °C , 00:10:00
g) 4 °C , hold
*Annealing Temp and Extension time will differ based on PCR design
3. After reaction, take 5 µL of product and add 1 µL of gel loading buffer and run on 1% agarose gel to make sure there is a correct PCR product.
4. Add 5 µL of Cutsmart buffer and 1 µL of DpnI to the reaction and incubate at 37 °C for 00:15:00 . This is remove the plasmid template from mixture.
5. Purify Band from gel with Promega Wizard SV Gel and PCR Purification Kit (A9282)
b) Weigh DNA gel fragment and add 10 µL of Membrane Binding Solution per 10 mg of gel slice.
c) Incubate mixture at 65 °C for 00:10:00 or until gel is completely melted.
d) Add melted gel mixture to SV minicolumn in Collection Tube and incubate at room temperature for 00:01:00 .
e) Centrifuge at max speed for 00:01:00 . Discard flowthrough and reinsert column into tube.
f) Add 700 µL of Membrane Wash Solution. Centrifuge at max speed for 00:01:00 . Discard flowthrough and reinsert column into tube.
g) Add 500 µL of membrane wash solution. Centrifuge at max speed for 00:01:00 . Discard flowthrough and reinsert column into tube.
h) Spin empty column for 00:01:00 at max speed to remove excess ethanol.
i) Transfer column to labelled 1.5 mL tube and add 35 µL of NF H20. Incubate for 00:01:00 at room temperature.
j) Centrifuge at max speed for 00:01:00 . Keep eluate and store at -20 °C .
47m