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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

QuikChange II XL Site-Directed Mutagenesis

• 5 uL 10x reaction buffer
• 1 uL plasmid template
• 1 uL Mutagenesis primer #1
• 1 uL Mutagenesis primer #2
• 3 uL QuikSolution
• 39 uL ddH2O
• Mix well, then add 1 uL PfuUltra HF DNA polymerase (keep on ice)

Mutagenesis PCR cycles:

1. 95°C for 2 minutes
3. Repeat following steps 30x

• 95°C for 20 seconds
• 55°C for 30 seconds
• 65°C for 6 minutes

4. 65°C for 5 minutes
5. Allow to set at 4°C and keep at that temperature

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

Thaw DH5α chemically competent cells on ice. Meanwhile, melt 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 2µl of the ligation mix to ~25µl 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 .5ml 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.

Colony PCR

One way to detect successful molecular cloning after transforming bacteria

• Set-up a standard 25µL Taq reaction mix using bacterial colonies as the template and primers that are specific to the plasmid and will amplify the insert.
• Label PCR tubes and new LB AMP plates, one of each per colony to be used.
• Carefully add 1 isolated colony to 50µL of water in a PCR tube and patch on labeled plate immediately.
• Boil for 5 minutes in the PCR machine. The DNA is now ready to use as a template.
• Set up a master mix of Standard TAQ PCR solution but cut in half for a 25µL reaction. Aliquot PCR solution to all tubes before adding template and create an extra reaction for a no template control.
• After running PCR (25 cycles should be sufficient), verify inserts by running 8µL of product on Standard 1% Agarose Gel only (not low-melt).

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 2µL ddH2O on the lens, follow instructions on the program.
• Clean lens with wipe and place 2µL of calibration buffer on the lens. Click the "blank" option.
• Clean lens with wipe and place 2µL of sample, then click "measure" in the program.