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

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
2. Repeat following steps 30x

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

3. 65°C for 5 minutes
4. 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.

Multiformis media preparation (from Ananda Shankar of the University of Utah)

1. Grow Multiformis nitrosospira at 280 C. Keep dark and do not shake.
2. Does not grow on plates
3. When you inoculate the media with Multiformis you should see a color change in the media within 1 – 1.5 weeks. This color change (pink to pale pink to yellow to white) indicates that the bacteria is growing. The bacteria needs to be kept at a pH of 7. You will know you need to adjust the pH when the color change occurs. To change the pH, add sodium bicarbonate (a carbon source for the bacteria). As growth occurs you will be able to see small flocks.
4. Media should not be stored and/or used for active growth of the bacteria for more than one month. To transfer to new media, pellet the cells down, discard supernatant and add fresh media.
5. Very sensitive to contamination as it is so slow growing
6. For growing up from glycerol stock, start in smaller container first and then work it into a larger container (500 mL).
7. If we do not see a color change within 2 weeks, contact Ananda.
8. Filter-sterilize sodium bicarbonate for use in adjusting pH when needed. Initial sodium bicarbonate can be autoclaved with the rest of the media. Media will appear yellow at first due to the heat of the autoclave, but will assume its pink color after it cools.
9. Ammonia will degrade; this is why it is important to change media. Also, this will prevent toxin buildup.
10. Multiformis will appear kidney shaped under a microscope, they are autotrophs
11. The culture he gave us is in media that is already about 3 weeks old, so we need to get Multiformis from the frozen stock growing as soon as possible and pellet the current culture and transfer into new media.
2.5 M (NH₄)₂SO₄ solution
• (NH₄)SO₄ 66.07 grams in 200 mL ddH₂O

EDTA(Fe) chelated iron

• A) Dissolve 2.62 grams of EDTA in about 50 mL of hot water (about 70^o C) containing 28.0 or 28.4 mL of 1 N KOH or 28.2 or 28.4 mL NaOH
• B) Dissolve 2.49 grams of FeSO₄*7H₂O or 1.78 grams of FeCl₂*4H₂O in about 30 mL of hot water (about 70^o C) containing 0.4 mL of 1 N H₂SO₄ (or HCl – see table, not sure which table he is referring to in his notes)
• C) Mix A and B and add water to about 90 mL, aerate vigorously for 1 hr, then make to 100 mL with water

Trace Elements Solution

• MnSO₄*H₂O 3.38 mg
• H₃BO₃ 4.94 mg
• ZnSO₄*7H₂O 4.31 mg
• FeSO₄*7H₂O 9.73 mg
• CuSO₄*5H₂O 2.50 mg
• (NH₄)Mo₇O₄ 3.71 mg
• Adjust to 100 mL with 0.01 N HCl

Phenol Red Solution

• 0.4 mg in 0.01 N NaOH

0.5 M Na₂CO₃ Solution

• 5.29 g in 100 mL ddHO

Add the following together:

• 2.5 M (NH₄₄ 5 mL
• EDTA(Fe) Sol. 1 mL
• Trace Element Sol. 1 mL
• Phenol Red 1 mL (add 0.3 mL-0.5 mL)
• MgSO₄ 0.096 grams (or 0.197 g MgSO₄*7H₂O)
• CaCl₂*2H₂O 0.015 grams
• K₂HPO₄*3H₂O 0.141 grams (or 0.087 g K₂HPO₄)
• Add ddb₂O to make up to 1000 mL, adjust the pH 7.2-7.4 with Na₂CO₃ and autoclave.
• *Filter sterilize 0.5 M Na₂CO₃ 5.29 grams in 100 mL ddH₂O for adjusting pH as bacteria grows.