TITLE: PCR and Gel electrophoresis/purification protocol
AUTHORS: Katie Jing Kay Lam, Claire D Clelland

[DESCRIPTION]
This protocol describes Polymerase chain reaction PCR, Gel electrophoresis and Gel purification.

[STEPS]
SECTION: DNA preparation by QuickExtract™
1. Wash cells (from a 96-well plate) with 100 µL PBS & aspirate



SECTION: DNA preparation by QuickExtract™
2. Add 30 µL QuickExtract™ and scrape well bottom with pipette tip to detach cells
SECTION: Primer preparation
8. Resuspend IDT primers with H2O to 100 µM
E.g. 20 nmol of primer (marked on tube label) - Add 200 µL H2O
SECTION: Primer preparation
9. Dilute primer to 10 µM in a new microcentrifuge tube
E.g. 20 µL + 180 µL H2O
SECTION: Primer preparation
10. Store at -20°C
SECTION: PCR reaction setup
11. Prepare Master Mix – Number of reactions + 1 (as extra)

Master Mix using BioMix Red - for one 20 µL reaction
 

Master Mix using BioMix Red - for one 20 µL reaction
 
 
SECTION: PCR reaction setup
12. For each PCR tube: 1 µL DNA sample + 19 µL Master Mix
SECTION: PCR reaction setup
13. Bio-Rad Thermal Cycler – Select/Edit Protocol

PCR Cycle
 

SECTION: DNA preparation by QuickExtract™
3. Transfer cells to labelled PCR tubes
Vortex for 15 secs
SECTION: DNA preparation by QuickExtract™
4. Incubate at 65°C for 6 mins
Vortex for 15 secs
SECTION: DNA preparation by QuickExtract™
5. Incubate at 98°C for 2 mins
SECTION: DNA preparation by QuickExtract™
6. Store at -20°C
SECTION: Gel electrophoresis preparation
14. Make 1x TAE buffer
Add 36 mL 50X TAE Buffer
Add diH2O up to 1800 mL
SECTION: Gel electrophoresis preparation
15. Gel preparation on Bio-Rad Sub-Cell GT Electrophoresis Cell system

 

SECTION: Gel electrophoresis preparation
15.1. Volume:
Big tray: ~ 300 mL
Small tray: ~ 150 mL
SECTION: Gel electrophoresis preparation
15.2. Gel percentage: 1.5-2%
E.g. For 2% gel: 2 g agarose + 100 mL 1X TAE buffer
SECTION: Gel electrophoresis preparation
15.3. Heat in microwave until completely dissolve
SECTION: Gel electrophoresis preparation
15.4. Add SYBR™ Safe DNA Gel Stain
E.g. 15 µL for 150 mL of gel
SECTION: Gel electrophoresis preparation
15.5. Pour onto the tray
Note: Remove big bubbles
SECTION: Gel electrophoresis preparation
15.6. Put in the comb (15/20 well)
SECTION: Gel electrophoresis preparation
15.7. Let it cool until solidify
SECTION: Running gel electrophoresis
16. Make sure the gel is solidified completely
SECTION: Running gel electrophoresis
17. Carefully remove comb from gel
SECTION: Running gel electrophoresis
18. Put tray into Bio-Rad Sub-Cell GT Cell
Note: Make sure the wells are placed near the end of the negative (black) terminal
SECTION: Running gel electrophoresis
19. Add 1X TAE buffer to the Sub-Cell GT Cell if needed
Note: Make sure the gel is submerged in TAE buffer completely
SECTION: Running gel electrophoresis
20. 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 

 


SECTION: Running gel electrophoresis
21. 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

 

SECTION: Running gel electrophoresis
22. START
SECTION: Gel imaging - LI-COR Odyssey M Imager
23. Position the gel in LI-COR Odyssey M Imager

 

SECTION: Gel imaging - LI-COR Odyssey M Imager
24. 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
SECTION: Gel imaging - LI-COR Odyssey M Imager
25. Wait for scan to finish
SECTION: Gel imaging - LI-COR Odyssey M Imager
26. Export image to desired destination
SECTION: Gel imaging - LI-COR Odyssey M Imager
27. Note: Remember to clean the imager glass with EtOH and Kimwipe before and after each use
SECTION: PCR for gel purification
31. For each PCR tube: 2.5 µL DNA sample + 47 µL Master Mix
SECTION: PCR for gel purification
30. Prepare Master Mix – Number of reactions + 1 (as extra)

Master Mix using BioMix Red – for one 50 µL reaction
 

Master Mix using Q5 – for one 50 µL reaction
 
 
SECTION: PCR for gel purification
32.     
Bio-Rad Thermal Cycler – Select/Edit Protocol
 
 
 
SECTION: Gel extraction -  modified from Qiagen MinElute Gel Extraction Kit[6]
33. Run gel electrophoresis as instructed above
SECTION: Gel extraction -  modified from Qiagen MinElute Gel Extraction Kit[6]
34. Excise the desired gel band under UV light
SECTION: Gel extraction -  modified from Qiagen MinElute Gel Extraction Kit[6]
35. Transfer the gel slice into a 1.5 mL tube
SECTION: Gel extraction -  modified from Qiagen MinElute Gel Extraction Kit[6]
36. Add 0.6 mL Qiagen Buffer QG
SECTION: Gel extraction -  modified from Qiagen MinElute Gel Extraction Kit[6]
37. Incubate at 50°C until gel has completely dissolved
Note: Mix the tube every 1-2 mins to help dissolve the gel
SECTION: Gel extraction -  modified from Qiagen MinElute Gel Extraction Kit[6]
38. Add 100 µL 100% isopropanol & mix by inverting
SECTION: Gel extraction -  modified from Qiagen MinElute Gel Extraction Kit[6]
39. Transfer sample to a MinElute spin column (stored at 4°C)
SECTION: Gel extraction -  modified from Qiagen MinElute Gel Extraction Kit[6]
40. Centrifuge at 8000 rpm for 1 min & discard the filtrate
Put back the spin column to the same collection tube
SECTION: Gel extraction -  modified from Qiagen MinElute Gel Extraction Kit[6]
41. Repeat previous two steps until all sample has passed through the column
SECTION: Gel extraction -  modified from Qiagen MinElute Gel Extraction Kit[6]
42. Add 650 µL Buffer PE to spin solumn
SECTION: Gel extraction -  modified from Qiagen MinElute Gel Extraction Kit[6]
43. Centrifuge at 8000 rpm for 1 min & discard the filtrate
Put back the spin column to the same collection tube
SECTION: Gel extraction -  modified from Qiagen MinElute Gel Extraction Kit[6]
44. Centrifuge at 13000 rpm for 1 min & discard the filtrate+collection tube
Place the column in a new 1.5 mL tube
SECTION: Gel extraction -  modified from Qiagen MinElute Gel Extraction Kit[6]
45. Add 35-50 µL Buffer EB
Note: Make sure it is added to the center of the membrane
SECTION: Gel extraction -  modified from Qiagen MinElute Gel Extraction Kit[6]
46. Incubate at 42°C for 3 mins
SECTION: Gel extraction -  modified from Qiagen MinElute Gel Extraction Kit[6]
47. Centrifuge at 13000 rpm for 1 min
SECTION: Gel extraction -  modified from Qiagen MinElute Gel Extraction Kit[6]
48. Repeat previous three steps to increase product yield
Note: Reload with the purified DNA product instead of adding new Buffer EB
SECTION: DNA preparation by QuickExtract™
7. 
 

SECTION: Gel imaging - LI-COR Odyssey M Imager
28. Quick-Load‱ Purple 1 kb Plus DNA Ladder

 

SECTION: Gel extraction -  modified from Qiagen MinElute Gel Extraction Kit[6]
49. 
 

SECTION: PCR for gel purification
29. Run a 50 µL reaction instead of 20 µL