Team:Austin UTexas LASA/Experiments
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.
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