Reactants

Protocol

1. On ice, add all reagents to PCR tube, Taq polymerase last.
2. Run thermocycler program:
• 95 C for 5 min (2 min if NOT using Hotstart)

> 10 cycles:
95 C for 20 s
0.3 C/s to 50 C
72 C for # s (extension = 1 min/kb)

> 15 cycles:
95 C for 20 s
55 C for 20 s
72 C for 1 min

72 C for 10 min
4 C forever

Reagents and protocol from Zymo DNA Clean & Concentrator Kit

Reagents

Protocol:all centrifugation steps should be performed between 10,000 - 16,000g.

1. On ice, add DNA Binding Buffer (2-7 volumes) and the sample (see table below) in a microcentrifuge tube and mix briefly by vortexing.

Application	DNA Binding Buffer: Sample	Example
Genomic DNA (> 2 kb)	2 : 1	200 uL : 100 uL
PCR Product, DNA Fragment	5 : 1	500 uL : 100 uL

2. Transfer mixture to a minicolumn inside of a collection tube and centrifuge for 30 seconds, discard flow-through.
3. Add 200 uL DNA Wash Buffer to the column and centrifuge for 30 seconds. Repeat 1x.
4. Add water (volume dependent on objective) directly to the column, and incubate at room temperature for 1 min.
5. Transfer the column to a clean microcentrifuge tube and centrifuge for 1 min to elute the DNA.

Reagents:

**Don’t forget ladder.
Protocol (gel):

1. Mix the TAE buffer and agarose once measured in microwavable flask. Stir with stir bar in flask and magnetic stirrer.
2. Afterwards, microwave for 1-3 mins until agarose is completely dissolved.
   Pause the microwave if the solution starts bubbling over, then stir the solution by slightly shaking the flask, and put the flask in the microwave again.
3. Let the flask cool until it is cool enough to carry with the orange heat “gloves.”
4. Add ethidium bromide and let gel solidify (10 - 20 min). Remember combs.
5. Usually, use the gel for gel electrophoresis right away, but if needed, the gel can be stored in a container filled with TAE buffer and 1-2 drops of ethidium bromide.

1. Add reagents together (always at least ladder and sample).
• This can occur in PCR tubes or parafilm.

# bp ladder - 2 uL
Loading dye - 4 uL (1:3.5 ratio)
Water - 8 uL

Sample - volume depends on objective. Remember: try to have at least 50 ng to visualize on gel.
Loading dye - 1:3 ratio with sample.

2. Place the gel in the gel tray in the gel electrophoresis chamber and fill with TAE buffer to max line.
   • Since DNA is negatively charged, the wells should be near the positive end (black) and DNA will “run to red.”
3. Put the ladder and sample(s) in the wells with the ladder in Well 1, etc. Close the chamber properly, and run the gel electrophoresis chamber at 100 - 130 V.
4. Wait for about 30 minutes before stopping the gel electrophoresis chamber and removing the gel tray.
5. Place the gel in the Gel Imager (UV Light) next to the computer that will show the image. Run the program “Ethidium Bromide” to see DNA bands.
   • Make sure to position the gel in the center.
   • Remember: smaller fragments are farther away from the wells.
6. Clean up area, making sure to throw the gel away in the gel waste bin.

Reagents

Protocol

1. Weigh a microcentrifuge tube for each DNA fragment to be isolated and record weight
2. Use a UV lamp to excise the DNA fragment of interest in a minimal volume of agarose using a clean scalpel or razor blade
3. Transfer the gel slice to the weighed microcentrifuge tube and record the weight
4. Subtract the weight of the empty tube from the total weight to obtain the weight of the gel slice
   • The gel slice may be stored at 4°C or at –20°C for up to one week in a tightly closed tube under nuclease-free conditions before continuing purification
5. Add Membrane Binding Solution to the gel slice
6. Vortex the mixture and incubate at 50–65°C for 10 minutes or until the gel slice is completely dissolved
7. Vortex the tube every few minutes to increase the rate of agarose gel melting
8. Centrifuge the tube briefly at room temperature to ensure the contents are at the bottom of the tube
   • Once the agarose gel is melted, the gel will not resolidify at room temperature
9. Transfer the dissolved gel mixture or prepared PCR product to the SV Minicolumn assembly
10. Incubate for 1 minute at room temperature before centrifugation for 1 minute and discarding supernatant
11. Wash with 700µL of Membrane Wash Solution, centrifuge for 1 minute, and discard supernatant
12. Wash with 500µL of Membrane Wash Solution, centrifuge for 5 minutes, and discard supernatant
13. Centrifuge 1 minute with the microcentrifuge lid open to allow evaporation of any residual ethanol
14. Transfer SV Minicolumn to a new microcentrifuge tube
15. Add Nuclease Free water directly to the center of the column
    • Membrane should be completely covered with nuclease-free water
16. Incubate at room temperature for 1 minute, centrifuge for 1 minute at 16,000 × g, and repeat
17. Discard the SV Minicolumn
18. Store the tube containing the eluted DNA at 4°C or –20°C
    • Check the purity of DNA in the nanodrop
    • If concentration not high enough, speed vac and nanodrop again

Reagents:

Procedure

1. Calculate the amount of insert, vector, and water needed. Use calculations on spreadsheets in drive.
   • 3:1 insert to vector ratio
   • Should have 500 ng of template DNA

Reagents	Concentration (ng/uL)	Concentration (g/uL)	# bp	Mol weight (amu)	M (Concentration/MW)	uL necessary for # fmol
Insert	given	Conc (ng/uL) * 10^(-9)	given	# bp * 650	Conc (g/uL) / mol weight	# fmol / M
Vector	given	Conc (ng/uL) * 10^(-9)	given	# bp * 650	Conc (g/uL) / mol weight	# fmol / M
Gibson Master Mix	n/a	n/a	n/a	n/a	n/a	15
NF H2O	n/a	n/a	n/a	n/a	n/a	20 - (sum)
				Total		20 uL

2. On ice, put the insert, vector, and water in a PCR tube with 15 uL of the gibson master mix.
   • There should already be aliquots of 15 uL of gibson master mix (GA) in the fridge. Restock if not.
3. Run thermocycler program.

50 C for 1 hr
4 C forever

Reagents

Protocol:

1. Calculate amount of reagents and DNA needed based on wanted volume of reaction and add all reagents to a PCR tube on ice, restriction enzyme last.
   • 20 fmol DNA (for each insert/part) per 10 uL rxn
2. Run thermocycler program.

37 C 1 hour
55 C 5 min

Reagents:

Protocol:

Reagents:

Equipment:

• Plate of Transformed Colonies
• Large Tubes in Tube Rack
• Bunsen Burner (sterile technique throughout)
• Shaking Incubator

Protocol:

1. In front of the flame from the bunsen burner, put the nutrient broth and antibiotic in the large tube
   • Superior broth has more nutrients than LB and therefore has a higher yield
   • Antibiotic depends on antibiotic resistance of transformed colonies
2. Pick up a colony from the plate of colonies by touching the colony with a tip (on pipette)
3. When transferring the colonies to the large tubes, tilt the large tube so the nutrient broth is closer to the opening
4. Put the very end of the tip (which touched the plate and has the colony) into the broth
   • Do not dip the entire tip in the nutrient broth because the tip is not that sterile
5. Shake the tip a little to transfer the colony into the nutrient broth
6. Take the very end of the tip out of the tube, discard the tube, and cap the tube
7. Put the tubes in a shaking incubator overnight
   • Can either miniprep immediately or freeze culture tubes to miniprep later

Freezing Liquid Culture Tubes

1. Take liquid culture tubes out of the shaking incubator and centrifuge at 3000 rpm for 5 minutes
2. Dump the supernatant (liquid) into a beaker
3. Add 10% bleach to the beaker
4. Place the large tubes in the freezer covered with plastic wrap
   • Take out the large tubes only when going to do the miniprep immediately

Purpose:
To purify plasmid from cells, usually with the liquid cultures created from a transformation

Reagents

Equipment:

• 1.5 mL Microcentrifuge Tubes
• Spin Column (with Collection Tube)
• Microcentrifuge

Protocol:

1. Pellet liquid culture in 1.5 mL microcentrifuge tube by centrifugation at 13000 rpm for 5-10 minutes, and discard the supernatant
   • Pipette the liquid culture into a labelled microcentrifuge tube 1 mL at a time
   • Centrifuge the 1 mL-filled tubes and discard the supernatant
   • Repeat this process, putting 1 more mL of the liquid culture into the same microcentrifuge tube, until the liquid culture tube is empty
   • Pellet should form inside the microcentrifuge tube
2. Resuspend the plasmid in 400 µL Plasmid Resuspension Buffer (B1)
   • Vortex or pipette until there are no clumps present
3. Add 400 µL Plasmid Lysis Buffer (B2), and gently invert the tube 5-6 times
4. Incubate at room temperature for 1 minute
5. Add 800 µL Plasmid Neutralisation Buffer (B3), and invert the tube until the entire solution is completely yellow
   • Plasmid Neutralisation Buffer (B3) should be stored in the 4 C freezer
6. Incubate at room temperature for 2 minutes
7. Centrifuge at 13000 rpm for 10 minutes
8. Transfer the supernatant into the spin column (with the collection tube)
9. Centrifuge at 13000 rpm for 1 minute, and discard the flow-through (in the collection tube)
10. Add 200 µL Plasmid Wash Buffer 1 to the spin column
11. Centrifuge at 13000 rpm for 1 minute, and discard the flow-through (in the collection tube)
12. Add 400 µL Plasmid Wash Buffer 2 to the spin column
13. Centrifuge at 13000 rpm for 1 minute, and discard the collection tube with the flow-through
    • If there is any doubt, centrifuge at 13000 rpm for 1 more minute before discarding the collection tube with the flow-through
    • Do not touch the spin column to the flow-through in the collection tube when removing the spin column
14. Transfer the spin column to a clean microcentrifuge tube
15. Add >= 30 µL Elution Buffer
    • Note: Using more elution buffer will increase overall yeild of DNA but will result in final solution of eluted DNA being less concentrated.
    • Revision: To increase the yield, use 20 uL of Michelle’s Zymo Elution Buffer instead
    • Revision: To decrease chemical contamination from ethanol, after step 13 spin the tubes with the filter for 1 min at 13,000 rpm without the lid in order to evaporate ethanol from the filters
    • Revision: To increase yield heat elution buffer using a heating block to 60℃
16. Incubate at room temperature for 1 minute
17. Centrifuge at 13000 rpm for 1 minute
    • Resulting flow-through in the microcentrifuge tube is the purified DNA

(Based on Monarch Miniprep protocol with revisions from Michelle Byrom)