Experiments
Reactants
10x Reaction Mix | 5 uL |
---|---|
For primer (10 uM) | 1 uL |
Rev primer (10 uM) | 1 uL |
Template | 1 uL |
Taq Polymerase | 1 uL |
MgCl2 (25 mM) | 1 uL |
DMSO | 1 uL |
NF H2o | 41 uL |
Total | 50 uL |
Protocol
- On ice, add all reagents to PCR tube, Taq polymerase last.
- 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
DNA Binding Buffer | Refer to table below. |
---|---|
DNA Wash Buffer | Refer to table below. |
NF H2O | Volume dependent on objective. |
Protocol:all centrifugation steps should be performed between 10,000 - 16,000g.
- On ice, add DNA Binding Buffer (2-7 volumes) and the sample (see table below) in a microcentrifuge tube and mix briefly by vortexing.
- Transfer mixture to a minicolumn inside of a collection tube and centrifuge for 30 seconds, discard flow-through.
- Add 200 uL DNA Wash Buffer to the column and centrifuge for 30 seconds. Repeat 1x.
- Add water (volume dependent on objective) directly to the column, and incubate at room temperature for 1 min.
- Transfer the column to a clean microcentrifuge tube and centrifuge for 1 min to elute the DNA.
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 |
Reagents:
TAE Buffer | ~ 50 mL per gel, more necessary for electrophoresis chamber. |
---|---|
NF Agarose (generally Seakem LE) | Grams of agarose : mL buffer = percentage of gel |
Ethidium Bromide | ~ 1 drop |
**Don’t forget ladder.
Protocol (gel):
- Mix the TAE buffer and agarose once measured in microwavable flask. Stir with stir bar in flask and magnetic stirrer.
- 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.
- Let the flask cool until it is cool enough to carry with the orange heat “gloves.”
- Add ethidium bromide and let gel solidify (10 - 20 min). Remember combs.
- 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.
Protocol (gel electrophoresis):
- Add reagents together (always at least ladder and sample).
- This can occur in PCR tubes or parafilm.
- 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.”
- 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.
- Wait for about 30 minutes before stopping the gel electrophoresis chamber and removing the gel tray.
- 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.
- Clean up area, making sure to throw the gel away in the gel waste bin.
# 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.
Reagents
Membrane Binding Solution | 1 mL per each g of gel |
---|---|
Membrane Wash Solution | 1200 uL |
NF water | 15 uL |
Protocol
- Weigh a microcentrifuge tube for each DNA fragment to be isolated and record weight
- Use a UV lamp to excise the DNA fragment of interest in a minimal volume of agarose using a clean scalpel or razor blade
- Transfer the gel slice to the weighed microcentrifuge tube and record the weight
- 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
- Add Membrane Binding Solution to the gel slice
- Vortex the mixture and incubate at 50–65°C for 10 minutes or until the gel slice is completely dissolved
- Vortex the tube every few minutes to increase the rate of agarose gel melting
- 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
- Transfer the dissolved gel mixture or prepared PCR product to the SV Minicolumn assembly
- Incubate for 1 minute at room temperature before centrifugation for 1 minute and discarding supernatant
- Wash with 700µL of Membrane Wash Solution, centrifuge for 1 minute, and discard supernatant
- Wash with 500µL of Membrane Wash Solution, centrifuge for 5 minutes, and discard supernatant
- Centrifuge 1 minute with the microcentrifuge lid open to allow evaporation of any residual ethanol
- Transfer SV Minicolumn to a new microcentrifuge tube
- Add Nuclease Free water directly to the center of the column
- Membrane should be completely covered with nuclease-free water
- Incubate at room temperature for 1 minute, centrifuge for 1 minute at 16,000 × g, and repeat
- Discard the SV Minicolumn
- 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:
DNA insert | 3:1 insert to vector ratio. Should have ~--- ng. |
---|---|
DNA vector | 3:1 insert to vector ratio. |
Gibson Master Mix | 15 uL |
NF H2O | Remaining volume necessary. |
Total | 20 uL |
Procedure
- 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
- 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.
- Run thermocycler program.
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 |
50 C for 1 hr
4 C forever
Reagents
DNA inserts/parts | |
---|---|
T4 Ligase Buffer 10X | |
T7 Ligase | |
Type II Restriction Enzyme (BsaI or BsmbI) | |
NF H2O | Remaining volume necessary. |
Protocol:
- 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
- Run thermocycler program.
37 C 1 hour
55 C 5 min
Reagents:
LB Broth | |
---|---|
Protocol:
Reagents:
Nutrient Broth | 4 mL |
---|---|
Antibiotic | 4 uL |
Equipment:
- Plate of Transformed Colonies
- Large Tubes in Tube Rack
- Bunsen Burner (sterile technique throughout)
- Shaking Incubator
Protocol:
- 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
- Pick up a colony from the plate of colonies by touching the colony with a tip (on pipette)
- When transferring the colonies to the large tubes, tilt the large tube so the nutrient broth is closer to the opening
- 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
- Shake the tip a little to transfer the colony into the nutrient broth
- Take the very end of the tip out of the tube, discard the tube, and cap the tube
- Put the tubes in a shaking incubator overnight
- Can either miniprep immediately or freeze culture tubes to miniprep later
Freezing Liquid Culture Tubes
- Take liquid culture tubes out of the shaking incubator and centrifuge at 3000 rpm for 5 minutes
- Dump the supernatant (liquid) into a beaker
- Add 10% bleach to the beaker
- 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
Plasmid Resuspension Buffer (B1) | 400 µL |
---|---|
Plasmid Lysis Buffer (B2) | 400 µL |
Plasmid Neutralisation Buffer (B3) | 800 µL |
Plasmid Wash Buffer 1 | 200 µL |
Plasmid Wash Buffer 2 | 400 µL |
DNA Elution Buffer | 40 µL |
Equipment:
- 1.5 mL Microcentrifuge Tubes
- Spin Column (with Collection Tube)
- Microcentrifuge
Protocol:
- 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
- Resuspend the plasmid in 400 µL Plasmid Resuspension Buffer (B1)
- Vortex or pipette until there are no clumps present
- Add 400 µL Plasmid Lysis Buffer (B2), and gently invert the tube 5-6 times
- Incubate at room temperature for 1 minute
- 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
- Incubate at room temperature for 2 minutes
- Centrifuge at 13000 rpm for 10 minutes
- Transfer the supernatant into the spin column (with the collection tube)
- Centrifuge at 13000 rpm for 1 minute, and discard the flow-through (in the collection tube)
- Add 200 µL Plasmid Wash Buffer 1 to the spin column
- Centrifuge at 13000 rpm for 1 minute, and discard the flow-through (in the collection tube)
- Add 400 µL Plasmid Wash Buffer 2 to the spin column
- 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
- Transfer the spin column to a clean microcentrifuge tube
- 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℃
- Incubate at room temperature for 1 minute
- 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)