Team:BYU Provo/Notebook/CommonProcedures
From 2014.igem.org
BYU 2014 Notebook |
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RedTAQ PCR (50 uL Reaction)
- 35ul ddH20
- 10ul 5X REDTAQ buffer (mix well before use!!)
- 1.0ul 10mM dNTP’s
- 1ul each Primer (50 uM stock)
- 1ul appropriate diluted template DNA
- Mix well then add 2.5ul REDTAQ polymerase
Set up reactions on ice, and keep them on ice until placing them on the PCR machine which has been pre-warmed to 94°C. Abundant templates only require 20-25 cycles for amplification; dilute/complex templates require 35-40 cycles. Extension times vary depending on target size.
- Standard PCR program:
- 95°C for 2 min
- 95°C for 30 sec
- 55°C for 30 sec
- 72°C for 2 min
- Repeat (2-4) 35 times
- 72°C for 5 min
- 4°C for forever
TAQ Polymerase PCR (25 uL Reaction)
- 2.5 uL Thermopol buffer
- 1 uL dNTPs
- 1 uL Forward Primer
- 1 uL Reverse Primer
- 18.5 uL ddH2O
- 1-2uL Template
- Add 0.25 uL TAQ Polymerase at the end right before you start the PCR reaction.
Q5 PCR Reaction (50 uL Reaction)
- 10uL 5x Q5 Reaction Buffer
- 1uL dNTPs
- 1uL Forward Primer
- 1uL Reverse Primer
- 10uL Q5 Enhancer
- 23.5uL ddH2O
- 1-2uL Template
- Add 0.5 uL Q5 Polymerase right before you start the PCR reaction.
Set up reactions on ice, making sure to keep Q5 Polymerase on ice until needed.
Phusion PCR
- 35ul ddH20
- 10ul 5X Phusion GC buffer
- 1.0uL DMSO
- 1.5ul 10 mM dNTP’s
- 1ul each Primer (50 uM stock)
- 1ul appropriate diluted template DNA
- 0.5ul Phusion Polymerase
- Standard Phusion PCR program:
- 95°C for 2 min
- 95°C for 30 sec
- 55°C for 30 sec
- 72°C for 2 min
- Repeat (2-4) 35 times
- 72°C for 5 min
- 4°C for forever
Standard 1% Agarose Gel
- 75ml of 1X TAE buffer and 0.75 grams of agarose (regular agarose, not low melt). Warm in microwave for about 60-90 seconds or until the agarose is completely dissolved.
- With gloves on, add 1 drop (~6ul of 1mg/ml) ethidium bromide and swirl to mix.
- Allow the flask to cool until the glass feels warm/hot, then pour into gel bed. Insert appropriate comb and allow to cool until solid.
Low-melt Agarose Gel
- Mix 0.75g of low-melt agarose with 75mL of TAE. Caution: Low-melt heats more quickly than standard agarose in the microwave.
- Add 5ul of ethidium bromide to the agarose solution before casting
- Use a large-tooth comb to form wells that will accommodate ~40-50µl of material.
Analysis of PCR Products by Agarose Electrophoresis
- Add 6uL of 10X loading dye (orange dye) to each 50ul PCR product.
- Move the gel into the proper orientation in the gel box. Cover your gel with 1X TAE buffer. Add 6uL of DNA reference ladder to the first well.
- Add 6uL of each PCR product into subsequent wells.
- Turn on the gel box power supply and run at 150-175 volts. It will take about 15-30 minutes to complete depending on the desired resolution.
- Gels can be visualized and recorded in the Alpha-Imager.
Restriction Digest of Plasmid Insert
- PCR product (insert) digestion (50 uL reaction)
- 6 µl 10X Cutsmart NEB buffer
- 50 µl (all of ) PCR product
(**always mix reagents well before adding enzyme as the final reagent)
- 1.5 µl of each restriction enzyme (PstI/HindIII)
After all of these are mixed together they must be placed in the 37ºC bath or incubator for at least 1 hour.
Restriction Digest of Plasmid Vector
- ~14 µl H2O
- 5 µl Cutsmart NEB buffer
- 20 µl DNA vector (pBAD, pLAT plasmid)
(**always mix reagents well before adding enzyme as the final reagent)
- 1.5 µl of each restriction enzyme (PstI/HindIII)
After all of these are mixed together they must be placed in the 37ºC bath or incubator for at least 1 hour.
Nano-Drop
Performing a nano-drop on your purified plasmid allows you to determine the concentration and purity of your plasmid
- Clean lens with kimiwipe
- Place 2 uL ddH2O on the lens, follow instructions on the program
- Clean lens with wipe and place 2 uL of calibration buffer on the lens. Click the "blank" option.
- Clean lens with wipe and place 2 uL of sample, then click "measure" in the program.