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From 2014.igem.org

BYU 2014 Notebook EDIT Procedures

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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:

1. 95°C for 2 min
2. 95°C for 30 sec
3. 55°C for 30 sec
4. 72°C for 2 min
5. Repeat (2-4) 35 times
6. 72°C for 5 min
7. 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:

1. 95°C for 2 min
2. 95°C for 30 sec
3. 55°C for 30 sec
4. 72°C for 2 min
5. Repeat (2-4) 35 times
6. 72°C for 5 min
7. 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.

Low-Melt Gel for Purification of Sticky-Ended Insert/Vector

The vector sample must have dye added so it sets down into the well.

• Add 6 µL of orange dye then load the entire sample in the low-melt gel.
• Run the gel at 90 volts (or else it may melt) for about 45 to 60 minutes.
• When the gel has run, observe under a portable hand-held long wavelength UV lamp to avoid DNA damage.
• Carefully remove the DNA bands from the gel with a razor blade. Place the DNA samples in clearly labeled microcentrifuge tubes.
• Cut out a slice of gel that has no DNA for use as a no-insert control sample (see Ligation section below).

Standard Ligation Reaction (15 µl)

Low-melt insert or vector gel slices should be heated to 65°C to liquify.

• 6.5µl H2O
• 1.5µl 10X ligase buffer (includes ATP)
• 1µl T4 DNA ligase
• 3µl vector
• 3µl insert (or no-insert control sample)

The first three components of this protocol may be combined as a master mix. Incubate these reactions at room temperature for at least 30 min.

Transformation into E.coli DH5α

Typically, E. coli strain DH5α is made chemically competent. Thaw DH5α chemically competent cells on ice. Meanwhile, melt the ligations for a few minutes at 65°C. Also, be sure you have 42°C heat block ready as well as LB-agar plates with the proper antibiotic. When the DH5α is thawed, quickly add 2 ul of the ligation mix to ~25 ul of competent cells. Flick or vortex the tubes briefly and put them back in the ice for 2-10 minutes (it is important that the DH5α be kept as cold as possible during this process). Heat shock at 42°C for 60 seconds. Immediately place the tubes back on ice for 2 minutes. Add .5 ml of plain LB to the reactions and incubate at 37°C for 30 minutes. Plate using glass beads and incubate at 37°C over night.

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.