Oct 02, 2024
PCR and Gel electrophoresis/purification protocol
- 1University of California, San Francisco
External link: https://clellandlab.ucsf.edu/protocols
Protocol Citation: Katie Jing Kay Lam, Claire D Clelland 2024. PCR and Gel electrophoresis/purification protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l22j3pl1y/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: January 25, 2024
Last Modified: January 25, 2024
Protocol Integer ID: 94167
Keywords: PCR, Gel electrophoresis, Gel purification
Abstract
This protocol describes Polymerase chain reaction PCR, Gel electrophoresis and Gel purification.
Materials
| A | B | C | |
| Reagent | Manufacturer | Catalog No. | |
| DNA preparation | |||
| QuickExtract DNA Extraction Solution | Biosearch Technologies | 76081-768 | |
| PCR | |||
| BioMix Red | Meridian Life Sceiences | C755F25 | |
| Dimethyl sulfoxide (DMSO) | Sigma Aldrich Fine Chemicals Biosciences | D8418 | |
| Gel electrophoresis | |||
| Quick-Load Purple 1 kb Plus DNA Ladder | New England Biolabs Inc. | N0550S | |
| SYBR Safe DNA Gel Stain | Invitrogen | S33102 | |
| TAE Buffer (Tris-acetate-EDTA) (50X) | Thermo Scientific | B49 | |
| A | B | |
| Equipment | Manufacturer | |
| PCR | ||
| Thermal Cycler | Bio-Rad | |
| Gel electrophoresis | ||
| LI-COR Odyssey M Imager | LI-COR | |
| PowerPac HC Power Supply | Bio-Rad | |
| Sub-Cell GT Horizontal Electrophoresis System | Bio-Rad |
DNA preparation by QuickExtract™
DNA preparation by QuickExtract™
Wash cells (from a 96-well plate) with 100 µL PBS & aspirate
Add 30 µL QuickExtract™ and scrape well bottom with pipette tip to detach cells
Transfer cells to labelled PCR tubes
Vortex for 15 secs
Incubate at 65°C for 6 mins
Vortex for 15 secs
Incubate at 98°C for 2 mins
Store at -20°C
Primer preparation
Primer preparation
Resuspend IDT primers with H2O to 100 µM
E.g. 20 nmol of primer (marked on tube label) - Add 200 µL H2O
Dilute primer to 10 µM in a new microcentrifuge tube
E.g. 20 µL + 180 µL H2O
Store at -20°C
PCR reaction setup
PCR reaction setup
Prepare Master Mix – Number of reactions + 1 (as extra)
Master Mix using BioMix Red - for one 20 µL reaction
| A | B | |
| BioMix Red | 10 µL | |
| DMSO | 0.6 µL | |
| Forward primer (10 µM) | 1 µL | |
| Reverse primer (10 µM) | 1 µL | |
| RNase/DNase-free H2O | 6.4 µL |
Master Mix using BioMix Red - for one 20 µL reaction
| A | B | |
| Q5 High-Fidelity 2X Master Mix | 10 µL | |
| Forward primer (10 µM) | 1 µL | |
| Reverse primer (10 µM) | 1 µL | |
| RNase/DNase-free H2O | 7 µL |
For each PCR tube: 1 µL DNA sample + 19 µL Master Mix
Bio-Rad Thermal Cycler – Select/Edit Protocol
PCR Cycle
| A | B | C | |
| Step | Temperature | Time | |
| Enzyme Activation | 94°C | 3 mins | |
| Denaturation | 94°C | 30 secs | |
| Annealing | 50-65°C | 30 secs | |
| Primary Extension | 72°C | 1 min/kb | |
| Repeat from step 2 (34x) | |||
| Secondary Extension | 72°C | 5 mins | |
| Hold | 4°C | ∞ | |
Note: Annealing temperature should be 5-10°C lower than Tm of primers
Gel electrophoresis preparation
Gel electrophoresis preparation
Make 1x TAE buffer
Add 36 mL 50X TAE Buffer
Add diH2O up to 1800 mL
Gel preparation on Bio-Rad Sub-Cell GT Electrophoresis Cell system
Volume:
Big tray: ~ 300 mL
Small tray: ~ 150 mL
Gel percentage: 1.5-2%
E.g. For 2% gel: 2 g agarose + 100 mL 1X TAE buffer
Heat in microwave until completely dissolve
Add SYBR™ Safe DNA Gel Stain
E.g. 15 µL for 150 mL of gel
Pour onto the tray
Note: Remove big bubbles
Put in the comb (15/20 well)
Let it cool until solidify
Running gel electrophoresis
Running gel electrophoresis
Make sure the gel is solidified completely
Carefully remove comb from gel
Put tray into Bio-Rad Sub-Cell GT Cell
Note: Make sure the wells are placed near the end of the negative (black) terminal
Add 1X TAE buffer to the Sub-Cell GT Cell if needed
Note: Make sure the gel is submerged in TAE buffer completely
Carefully load DNA ladder & PCR products
Notes:
- Submerge pipette tips into the well before dispensing PCR product
- Prevent touching the wall of wells, which might break the gel
- Recommend dispensing liquid by pressing to the first stop only to prevent creating air bubbles, which could lead to loss of PCR product
Set 100-140V on the Bio-Rad Power Supply
Notes:
Voltage depends on gel size
Make sure the gel is connected to the power supply properly - Black to Black; Red to Red
START
Gel imaging - LI-COR Odyssey M Imager
Gel imaging - LI-COR Odyssey M Imager
Position the gel in LI-COR Odyssey M Imager
Open LI-COR Acquisition Software
Select:
- Scan
- Username: ________
Imager: Odyssey M --> Connect
- Gel --> Connect
- Draw Scan Area --> Next
- 488 SYBR Safe --> Save
- Focus offset (mm): 2.00 --> Scan
Wait for scan to finish
Export image to desired destination
Note: Remember to clean the imager glass with EtOH and Kimwipe before and after each use
Quick-Load‱ Purple 1 kb Plus DNA Ladder
PCR for gel purification
PCR for gel purification
Run a 50 µL reaction instead of 20 µL
Prepare Master Mix – Number of reactions + 1 (as extra)
Master Mix using BioMix Red – for one 50 µL reaction
| A | B | |
| BioMix Red | 25 µL | |
| DMSO | 1.5 µL | |
| Forward primer (10 µM) | 2.5 µL | |
| Reverse primer (10 µM) | 2.5 µL | |
| RNase/DNase-free H2O | 16 µL |
Master Mix using Q5 – for one 50 µL reaction
| A | B | |
| Q5 High-Fidelity 2X Master Mix | 25 µL | |
| Forward primer (10 µM) | 2.5 µL | |
| Reverse primer (10 µM) | 2.5 µL | |
| RNase/DNase-free H2O | 17.5 µL |
For each PCR tube: 2.5 µL DNA sample + 47 µL Master Mix
Bio-Rad Thermal Cycler – Select/Edit Protocol
| A | B | C | |
| Step | Temperature | Time | |
| Enzyme Activation | 94°C | 3 mins | |
| Denaturation | 94°C | 30 secs | |
| Annealing | 50-65°C | 30 secs | |
| Primary Extension | 72°C | 1 min/kb | |
| Repeat from step 2 (39x) | |||
| Secondary Extension | 72°C | 5 mins | |
| Hold | 4°C | ∞ | |
Note: Annealing temperature should be 5-10°C lower than Tm of primers
Gel extraction - modified from Qiagen MinElute Gel Extraction Kit[6]
Gel extraction - modified from Qiagen MinElute Gel Extraction Kit[6]
Run gel electrophoresis as instructed above
Excise the desired gel band under UV light
Transfer the gel slice into a 1.5 mL tube
Add 0.6 mL Qiagen Buffer QG
Incubate at 50°C until gel has completely dissolved
Note: Mix the tube every 1-2 mins to help dissolve the gel
Add 100 µL 100% isopropanol & mix by inverting
Transfer sample to a MinElute spin column (stored at 4°C)
Centrifuge at 8000 rpm for 1 min & discard the filtrate
Put back the spin column to the same collection tube
Repeat previous two steps until all sample has passed through the column
Add 650 µL Buffer PE to spin solumn
Centrifuge at 8000 rpm for 1 min & discard the filtrate
Put back the spin column to the same collection tube
Centrifuge at 13000 rpm for 1 min & discard the filtrate+collection tube
Place the column in a new 1.5 mL tube
Add 35-50 µL Buffer EB
Note: Make sure it is added to the center of the membrane
Incubate at 42°C for 3 mins
Centrifuge at 13000 rpm for 1 min
Repeat previous three steps to increase product yield
Note: Reload with the purified DNA product instead of adding new Buffer EB
Protocol references
- Biosearch Technologies. QuickExtract DNA Extraction Solution. Retrieved May 25, 2023, from https://cdn.media.amplience.net/i/biosearchtech/Epicentre-i_qexpcrreadydna.gif?sfvrsn=0.17608973563178687
- Bio-Rad. Sub-Cell GT Cell. Retrieved May 25, 2023, from https://www.bio-rad.com/sites/default/files/styles/brc_pdp_470x302_/public/webroot/web/images/lsr/products/electrophoresis/product_detail/global/lsr_sub_cell_gt_cell.webp?itok=XEdPGEIY
- Figure 4, and part of Figure 3, Figure 7 are created with BioRender.com
- LI-COR. Odyssey Imagers. Retrieved May 25, 2023, from https://www.licor.com/images/bio/photos/odyssey-m/odyssey-m-studio-facing-left.png
- New England Biolabs. Quick-Load‱ Purple 1 kb Plus DNA Ladder. Retrieved May 25, 2023, from https://www.neb.com/products/-/media/catalog/gel-photos/n3200_thumb.gif
- Qiagen. MinElute Gel Extraction Kit. Retrieved May 25, 2023, from https://www.qiagen.com/us/products/discovery-and-translational-research/dna-rna-purification/dna-purification/dna-clean-up/minelute-gel-extraction-kit?catno=28604