User:AgiStachowiak/S10NilesModTemp

S10 Niles Module: Methods

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


 * 1) In the meantime, …

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: ).

In vitro transcription

 * 1) The recommended DNA amount is 0.2-2 &mu;g per 40 &mu;L 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 &deg;C heat block. After 3-4 hours, they will be frozen at – 20 &deg;C until next time.

OR


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

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 &mu;L 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 &mu;L) 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 &mu;L.
 * 2) Add 100 &mu;L 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 &mu;L 70% EtOH (1 min spin).
 * 6) Repeat, then dry for 5 min in the hood, and resuspend in X &mu;L SB.

Buffer equilibration and column preparation

 * 1) Start by preparing the negative selection beads. Put 200 &mu;L 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.
 * 6) *Don’t throw away the tip!

MEASURE RNA CONCENTRATION BEFORE AND AFTER?

RNA aptamer preparation

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

SELEX column preparation and selection
(#Maybe repeat once or twice, maybe not at all.)
 * 1) Wet the column with 200 &mu;L 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 &mu;L 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 &mu;L 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.

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 &deg;C O/N.
 * 3) Next day: spin 15 min at max speed.
 * 4) Wash twice with 1 ml EtOH (2 min spin each).
 * 5) *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.
 * 6) Dry for 10’, then resuspend in 44 &mu;L 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 &mu;L of Master Mix to 20 &mu;L 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.

Binding assay

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