Team:iTesla-SoundBio/PCRPCRPurificationandAnalyticalGelProtocolTemplate

DON’T EDIT HERE, EDIT IN THE LAB NOTEBOOK!

Red text are things to keep in mind that you may or may not need to include in the actual lab notebook (After copying template over to lab notebook, delete any red you don’t need).

Highlighted text are things that you need to fill in or change for your particular lab notebook depending on the variables, and concentrations.

 

PCRing FC 1, PCR Purification, and an Analytical Gel:

 

Making new working solutions of primers (Only need to do this if there isn’t already a 10uM working solution:Prepare a 10uM working solution by creating a 10x dilution by adding 9 ul of ddH2O with 1ul of primer in a separate PCR tube. Label accordingly, gently flick the tube, then spin down in a mini-centrifuge for a few seconds.

 

Materials:

-PCR tubes and tube racks [storage]

-reverse primer working solution [          ]

-forward primer working solution [           ]

-RNA-ase free H2O [purple rectangular box in Sniffles I]

-Phusion Buffer 5x HF  [Sniffles purple rectangular box]

-Phusion Polymerase [Sniffles, “Sound Bio Enzymes” box}

-Gblock to PCR [               ]

-dNTPs [Sniffles, “Sound Bio Enzymes” box]

-ddH2O [laminar flow hood]

 

Procedure:

***Keep buffer, dNTPs, and polymerase on ice throughout procedure (or take it out of the freezer only when you’re about to use it).

2. Label  PCR tubes:
3. Pipette 35.5 ul of RNase-free water into labeled PCR tube. Make sure to pipette all following reagents into this water bubble to ensure the whole sample is expelled.
4.  Pipette 10 ul of Phusion Buffer into tube.
5. Pipette 1 ul of 10uM forward primer into tube
6. Pipette 1 ul of 10uM reverse primer into tube
7. Pipette 0.5 ul of appropriate geneblock into the relative tubes
8. Pipette 1 ul of dNTPs into tube.
9. Pipette 1 ul of Phusion Polymerase into tube
10. Centrifuge PCR tube in the mini PCR tube centrifuge for five seconds.
11. Place PCR tubes in Sleepy. Record below where each tube is placed in case the label wears off: ______________

 

13. Run the program applicable for your gblock PCR. For example, for FC 1, the thermocycling  protocol used is from Subdirect: Standard, Name: GENERAL. As a whole, NEB Phusion thermocycling conditions should look something like this: (taken from https://www.neb.com/protocols/1/01/01/pcr-protocol-m0530

And

https://www.k-state.edu/hermanlab/protocols/StandardPCRConditions.html

STEP	TEMP	TIME
Initial Denaturation	98°C	30 seconds
25-35 Cycles (Annealing, Extending)	98°C 45-72°C (5 degrees below Tm of primers) 72°C	5-10 seconds 10-30 seconds 15-30 seconds per kb
Final Extension	72°C	5-10 minutes
Hold	4-10°C

 

PCR Purification Procedure: (If using Qiagen columns, use 1 wash of Buffer PE instead of these instructions. Follow these if using GeneCatch column )

 

Materials:

-Four microcentrifuge tubes [back storage]

-Collection tubes and spin columns [Qiagen kit under bench 3]

-Buffer PB [Qiagen kit under bench 3]

-Buffer PE [^^^]

-Elution Buffer [^^^]

-PCR’d gblock [from last step]

From

https://www.qiagen.com/us/resources/resourcedetail?id=3987caa6-ef28-4abd-927e-d5759d986658&lang=en

1. Label ___ microcentrifuge tubes _________
2. Pipette all liquid from PCR solution into the microcentrifuge tubes.
3. Pipette 5 volumes of Buffer PB into microcentrifuge tubes. Pipette up and down to mix.
4. Set up and label a spin column in a collection tube for each PCRed solution
5. Pipette all the mixture from the microcentrifuge tubes into the spin columns.
6. Centrifuge columns at 17,900 rcf for 60 seconds. Dispose of flow-through.
7. Add 750 uL of Buffer PE to wash
8. Centrifuge columns at 17,900 for 60 seconds. Dispose of flow-through.
9. Centrifuge again for another 1 min to remove residual ethanol.
10. Move spin columns onto a new set of microcentrifuge tubes. Discard collection tubes.
11. Pipette 30 ul of Elution Buffer onto the center of each spin column (make sure the liquid is on the membrane). Let columns sit for four minutes.
12. Centrifuge columns at 17,900 for two minutes.
13. Dispose of spin columns. Record where samples and all other materials are put back. ____________

 

Nanodropping the Purified PCR:

 

Using the Nanodrop:

1. Plug NanoDrop USB into computer.
2. Open NanoDrop application (maybe?)
3. Use a p200 to put some amount of clean water on a wipe, then use said wipe to wipe off the black circle and the corresponding part on the lid. Keep the wipe around for reuse.
4. Pipette 0.65 uL of Elution Buffer into the middle of the black circle on the NanoDrop machine, the n slowly close the arm.
5. Press “Blank” (in the application) to blank the NanoDrop (this gives it a baseline from which to measure your sample’s concentration).
6. After it’s finished blanking, wipe the NanoDrop off with the wipe from earlier.
7. Pipette 0.65 uL of [whatever is being measured] into the NanoDrop as before. If you’ve just done the miniprep or taken the tubes out of the freezer, make sure to flick them a few times to a more consistent concentration. Close the lid.
8. Label the sample in the “Sample ID” box.
9. Press “Measure.”
10. Get the result. The graph should (hopefully) look something like this:
11. Take a screenshot and save it, naming it like so: [sample name] [today’s date]
12. Open the NanoDrop Results application from the desktop and enter the relevant information.

 

Results:

Label	Concentration	Volume	Location	Notes

Analytical Gel for PCR’d frag one and Phusion control:

 

Materials:

-TAE Solution [carboy near sink]

-250mL flask [SoundBio storage]

-Loading Dye [gel cart by bench 1]

- 20,000X Gel stain [foil envelope in Chilly]

-Parafilm [next to microwave]

-scale and weigh boat

-Graduated cylinder [storage]

-gel comb, gel tray (10 wells) [gel cart by bench 1]

-power supply [gel cart]

-PCR products [from last PCR]

 

Procedure:

1. Measure out 30mL TAE in a graduated cylinder.
2. Measure out 0.3 g of agarose using a weigh boat and the the scale
3. Pour 15mL from graduated cylinder into the flask.
4. Add the 0.3 g of agarose into the flask, and add rest of the TAE.
5. Microwave flask for 30 secs, and then microwave in 10 sec intervals until there is no more agarose visible, and it hasn’t started bubbling. Make sure to place some paper towel in the open end of the flask so it doesn’t bubble over.
6. Allow flask to cool, swirling with a glove every couple minutes to prevent solidification.
7. Pipette 1.5 uL of 20,000X gel stain into the cooled flask.
8. Pour contents of flask into gel tray until it reaches the tip of the gel tray to avoid flowover and wait for it to solidify (~20 minutes). Tape the ends of the gel tray to prevent the solution from flowing out. Remove tape and gel comb after solidification.
9. Place gel tray in gel box and fill gel box with TAE solution until gel tray is submerged.
10. Pipette the following reagents (in table) onto parafilm in the order they will appear on the gel. Pipette up and down to mix. Take care to remember which tube corresponds to which droplet.
11. Load from parafilm into wells using the following diagram:

1	2	3	4	5	6	7	8
3 ul 1 kb ladder	3uL purified PCR sample + 2uL LD (insert PCR sample name)

 

12. Connect to power supply and turn it on at 140 V
13. Wait until purple band is ½ of the way down the gel and then shut off
14. Once gel has finished running, view it under the blue light reader.

 

[Insert Picture of Gel]