User:AgiStachowiak/S10NilesModTemp

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S10 Niles Module: Methods

Contents

D1?

Amplify DNA fragment encoding aptamer

  1. You will be given plasmids encoding the DNA for two aptamers: “6-5” (at X μg/mL) and “8-12” (at X μg/mL). Make a plan for adding 2 ng of DNA to each PCR reaction, in no more than 20 μL final volume of plasmid and water.
  2. Per reaction, add 20 μL of Master Mix, 5 μL of each primer, the DNA template, and water to a total of 50 μL.
  3. The PCR will run for about 1 hour, on the following cycle:

TABLE

  1. In the meantime, …

PCR exercise?

Prepare agarose gel for next time

Begin SELEX buffer preparation

D2?

Run PCR products on gel

  • 3% gel with 2:1 Hi-Res to regular agarose works nicely

=== Purify PCR products

  • Qiagen kit

===Begin SELEX column preparation

  • Or will just use Sigma hemin-agarose beads? Getting enough MPIX coupling has been an issue

====Prepare MPIX solution

  1. In the fume hood, pipet 1 mL of DMSO into a clean eppendorf tube.
  2. Using the thin spatula provided, transfer a small dab of MPIX into the DMSO.
  3. Prepare a 1:1000 dilution of your stock in 40% DMSO (aqueous).
  4. Measure the absorbance of the diluted solution at 394 nm, and calculate your stock concentration. The extinction coeefficient for MPIX is 170 cm/mM in 40% DMSO (Source: ).


D3?

In vitro transcription

  1. The recommended DNA amount is 0.2-2 μg per 40 μL reaction, with 16.4% of the reaction volume available for the DNA. Begin by calculating how much [Prev FNT] DNA you can add.
  2. Per reaction – aptamer 1, aptamer 2, (and no T7 control?), use:

TABLE (20 uL rxn)

Rxn buffer 8

1 N KOH 1.12

Pyrophosphatase 1

NTPs 5.6

T7 1

DNA/H2O 3.28

  • I have been running 80 uL rxns, as this maps to the capacity of the Micro Bio-Spin columns
  1. Put your reactions in the 37 °C heat block. After 3-4 hours, they will be frozen at – 20 °C until next time.

OR

  1. Put your reactions in the 37 °C heat block. When everyone is ready, we will have today’s lecture and quiz. After 3 hours, add 2 (per 20) μL DNase I to each reaction. After 30 min, they will be frozen at – 20 °C until next time.

D4?

Purify IVT reaction

  1. Take one Micro Bio-Spin Columns and one 2 mL microfuge tube per reaction, and for each:
  2. Rapidly flip the column back and forth to resuspend the gel, then flick or tap it to rid air bubbles.
  3. Snap off the plastic tab at the bottom of the column (a little buffer may flow out), and place the column in the 2 mL microfuge tube. Let the excess buffer drain off.
  4. When the buffer stop flowing, centrifuge the tube for 2 min at 1000 g. (Is g rcf or rpm? Ask if you’re not sure!)
  5. Add 500 μL of RNase-free water [OR SELECTION BUFFER? - 99% yes, column dries more completely] to the top of the column, then [LET DRAIN OR] spin for 1 min at 1000g.
  6. Repeat 3 more times. Meanwhile, label 1.5 mL eppendorf tubes for collection (on the side and on the cap), and cut off their caps.
  7. Transfer each column to a 1.5 mL tube – both column and tube should be labeled, to be on the safe side – then add your IVT reaction (10-75 μL) gently to the center of the top surface of the gel.
  8. Centrifuge for 4 min, at 1000 g. Keep on ice for now… or -80 right away…

Alternative purification

  1. Dilute IVT rxn in water to 100 μL.
  2. Add 100 μL of chilled 5M NH4Ac.
  3. Incubate on ice (at -20?) for a minimum of 15 min (max O/N?)
  4. Spin at 10,000 rcf for 15 min (longer?)
  5. Remove the supernatant, then wash with 500 μL 70% EtOH (1 min spin).
  6. Repeat, then dry for 5 min in the hood, and resuspend in X μL SB.

D5?

Buffer equilibration and column preparation

  1. Start by preparing the negative selection beads. Put 200 μL of 2X slurry into an eppendorf tube, spin for 1 min at 1000 rcf, then remove the supernatant.
  2. Add a 10X resin volume of selection buffer and put on the nutator for 1 min.
  3. Allow the resin to settle, pipet off the supernatant, and repeat.
  4. Do steps 1-Y in the section below, then prepare the positive selection beads during the 30 min incubation in step Y, just as you did in steps 1-3 above.
  5. Stabilize an empty Bio-Rad column using a ringstand.
    • Don’t throw away the tip!

MEASURE RNA CONCENTRATION BEFORE AND AFTER?

RNA aptamer preparation

  1. Set aside X μL of the RNA mixture and label it “pre-selection.”
  2. Heat X μL of the remaining RNA mixture (200-1000 pmol) at 70 °C for 5 min.
  3. Add an equal volume of 2X selection buffer, then add 1X selection buffer (SB) to a total volume of 200 μL.
  4. Add the sample to 100 μL negative selection resin. Place on the nutator for 30 min.
  5. Remove the supernatant (~ 200 μL) and place in a new eppendorf tube.
  6. Optional? Wash the negative selection resin with 100 μL SB, then add to the previous supernatant.
  7. Incubate the total supernatant (~300 μL) with 200 μL of positive selection resin your prepared for 1 hour, on the nutator.

SELEX column preparation and selection

  1. Wet the column with 200 μL of SB, and let it drain.
  2. Add the positive selection resin/aptamer mixture to the column, and let drain.
  3. Now you must rinse non-binding materials from the column. Start by adding 200 μL of SB to the top of the column, and allow it to drain.
  4. Repeat X times, according to the table below… (or have students sign up for different wash numbers, or perhaps other variations on the protocol as well)
  5. Finally, plug up the column, and add 200 μL of 2.5 mM hemin, which will be used to elute the aptamers. Incubate for 10 min.
  6. Allow the eluant to drain by gravity into a well-labeled eppendorf.

(#Maybe repeat once or twice, maybe not at all.)

HAVE THEM SAVE SAY EVERY 3rd WASH TO CHECK 260/280 afterward?

Aptamer purification

  1. Add 1/10X volume of NH4Ac, 1/200X glycogen, and 2.5X 100% ethanol, in that order.
  2. Put at -20 °C O/N.
  3. Next day: spin 15 min at max speed.
  4. Wash twice with 1 ml EtOH (2 min spin each).
    • Note that your pellet may have a slight brownish hue from the large excess of heme – this should all be diluted out in the next steps.
  5. Dry for 10’, then resuspend in 44 μL water.

RT-PCR

HAVE THEM DO THE CONTROL I DID, OF MAKING SURE 6-5 AND 8-12 AMPLIFY AT SAME RATE?

  1. Add 30 μL of Master Mix to 20 μL of RNA template. Reactions will be run with the following thermocycling parameters.

TABLE

PROBABLY WILL RUN OUT ON GEL BUT NOT TAKE TIME TO PURIFY

  • Quantify on spec: 260, 280
    • Have to make assumption about 8-12:6-5 recovery
    • MW 31344 6-5, 33824 8-12

Students will test the pre-SELEX aptamer blend, the post-SELEX aptamer blend, and pure 6-5 and 8-12 via porphoryin binding assay.

Post-SELEX IVT

  1. Repeat the day X protocols (takes 2 days, do part for them? OR JOURNAL CLUB DAY) then quantify your RNA.

D6?

Binding assay

  1. Based on spec. quant, prepare 1.4 nmol aptamer/175 μL selection buffer
  2. Heat 5 min at 70 °C, then cool
  3. Meanwhile, prepare 6 μM heme/175 μL selection buffer
  4. Combine the aptamer and heme and incubate 5 min, then measure the 350 μL solns on the spec
    • From 350-425 nm, 0.1 nm at a time
    • Save on USB as you go!
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