PQQ Aptamers Ordered (IDT):

Aptamer 2: (15ADb11): /5Biosg/GA ACT AGA TCG CAG CCC CAC AGT CGA GAA GCA GTA AGA CGA CAT GGG AAC AAA CAT GGG GCC AAG AGA TCT AGG GCA CGC TGT GTG GAT CGA GGT AAT CC

Made B&W Buffer

Realized this week that we overlooked a fundamental problem in our protocol: we neglected to acknowledge the fact that PCR amplification will provide us with a double-stranded product, and we need to be able to separate out the desired single stranded DNA sequenced selected for in the capture-SELEX washes. We looked back to the literature and realized that we need to run a denaturing PAGE gel. We also need to order a special primer with a hexa-ethyleneglycol spacer and polyA tail that will help us separate out the "sense" strand from the "antisense" strand.

Today we ordered a modified primer for the intermediate PCR amplification steps: AAAAAAAAAAAAAAAAAAAAAAAAAAAAAA/iSp18/GATTGCACTTACTATCT

It will take at least a week to arrive.

We still need to order the proper denaturing gel supplies.

Made 1M CuSO₄ stock

Washed Magnetic Streptavidin Dynabeads

Dissolved and heated Supplemental PQQ to make ~10 uM stock to compare with 10 uM Pure PQQ Stock

Immobilized Biotinylated Oligonucleotides on beads (30 min incubation)

1:1:1 ratio of beads to aptamer to PQQ was added (shaking overnight in Binding Buffer) --> in theory, once bound these should still be catalytically active

PQQ Experimental Layout (see: http://pubs.rsc.org/en/Content/ArticleLanding/2015/RA/c4ra11052h#!divAbstract)

Made Rxn Buffer (500 uL 1M Lysine, 500 uL 1M CuSO4₄, 9 mL Phosphate Buffer), and distributed 100 uL per well

100 uL per well of respective PQQ/Aptamer Combination (see column A above) to achieve a target volume of 200uL

1:10 serial dilutions were performed from left to right of the aptamer-PQQ catalytic beads

PQQ acts as a co-factor for horseradish peroxidase, so we expect a characteristic pink color to be formed.

After a long time, the reactions in each well did indeed go to completion

Once plate setup was, added 100x amplex red to each well, and waited 30 minutes for dynamic reaction to occur (all wells expected to go to completion), then imaged immediately. Here is the raw data:

Made Selection Buffer, TE Buffer, TBE Buffer, more B&W Buffer

Washed bead complexes + DNA, washed with pABA

Collected all washes and labeled all of them, just in case

Collected flow-through, set aside 10% in tube labeled "Selex rd. 1"

Used remainder of flow-through for PCR amplification!

Thermocycler settings:

98C (30s)

[98C (10s)

50C (20s)

72C (20s)] x30

72C (2min)

4C (infinity)

Pooled and precipitated DNA with ethanol in presence of linear polyacrylamide

PCR product pre-gel: 1557.6 ng/uL in about 300uL

Today, we hit another wall, as we attempted to figure out how to properly visualize the DNA strands.

We had previously realized that we could not use an ordinary gel extraction kit, since our DNA strands were too small and would likely be lost in the filtration process. We researched various gel extraction kits designed for short nucleotide sequences, and all of their DNA loss rates were unacceptable for our purposes.

Mark Ditzler at NASA Ames told us his strategies for extracting RNA from denaturing gels using UV shadowing. Unfortunately, we realized that we do not have the proper UV lamp (254nm) with which to do this. Apparently these lamps are infrequently used because they damage DNA.

We have a new idea for DNA visualization ("borrowed" from Kosuke at Ames): After running the gel, we'll use GelRed to stain a lane with the DNA ladder + a lane with a small DNA sample. We'll then line up the stained gel against the unstained gel, and we'll use the stained gel as a template to approximate the location of the desired DNA band. We'll then mark the gel and extract the parts we want.

Afterwards, we can stain the non-extracted gel to confirm that we extracted the bands we meant to extract.

We ran a gel: one ladder, 2 lanes with 1uL PCR product (stained/unstained), 2 lanes with 10uL PCR product (stained/unstained)

After staining, we imaged the gel using a UV filter, and we could see two distinct bands:

After this successful gel run, we determined that we could safely use 10uL per well in future runs.

We ordered proper materials for this gel, to avoid future complications: sampl loading dye, 10bp DNA ladder for denaturing gel

Ran another gel, this time with 2 lanes of 10uL (stained/unstained) + 2 ladders (stained/unstained)

Followed Mini-PROTEAN TBE-Urea 10% Precast Gel Protocol

Followed protocol to make ssDNA ladder:

To 5uL of 10bp DNA ladder (1ug/uL), add equal volume denaturing soln (95% formamide, 10mM EDTA (pH 8.0), 0.1% bromophenol blue, 0.1% xylene cyanol)

Incubate @ 70C for 5min

Electrophorese sample in denaturing gel, 2ug/lane

Well proportions: 5uL ladder + 5uL sample buffer, 10uL DNA + 10uL sample buffer

Ran gel at 200V for about 65min, until the dye just ran off the bottom (remember, our DNA strands are tiny)

After staining half the lanes and placing under blue light to confirm proper separation, we used the stained gel as a template to extract the correct DNA band

Using our post-extraction staining method, we confirmed that we had successfully extracted a piece of unstained gel w/ the desired DNA band!

Ran rest of DNA in one gel, 10uL DNA per lane

Used 1uL DNA ladder, 10uL DNA + 10uL SB

Because we didn't want to waste DNA, we decided to use the leftover DNA +sample buffer stock from yesterday as our sample to stain. (In retrospect, this was a very bad call.)

Ran at 200V for 45min, then continued to watch gel until dye just ran off, as yesterday. Total of around 65min.

This time, the sample/DNA ladder ran faster than the DNA in the other wells.

When we used the same extraction method as yesterday, we missed the vast majority of the DNA. We hope that we got enough to move onto the next round.

We realized our mistake when we stained the remaining non-extracted gel and saw the DNA bands we meant to have extracted. (The ladder did not provide sufficient guidance, we realized, because with nucleotide strands this small, base pair composition affects migration in a significant way.)

We cut out the stained bands and saved them as well, just in case we ultimately need to salvage DNA. We would need to find a way to get rid of the stain in order to move forward with this DNA.

Added sodium acetate solution to DNA, followed extraction protocol (listed in our Capture SELEX protocol) as determined through studying the literature

Achieved heating step by placing 15mL tube on rotator in the oven.

(In retrospect: Mark Ditzler at Ames says he does not heat as the authors of our model paper do, but he does leave the gel + DNA solution incubating overnight. Perhaps we can refine this step in the protocol.)

Transferred the DNA into 1.5mL centrifuge tube, performed precipitation step with ethanol in presence of linear polyacrylamide

16 tubes

300uL sodium acetate soln

750uL ethanol

3.5uL LP

Centrifuged at max (13.3rpm) for 45min

Did this for stained gel and unstained gel extractions, labeling each separately. There are a lot of tubes.

Did not have time to nanodrop all the tubes, will do so when next in lab.

School started, and my recording became disorganized. Last week I came in at some point (perhaps 10/1?) and nanodropped all the tubes that should have contained precipitated DNA. None of them had DNA in them, according to the Nanodrop 2000. Keeping in mind that we had high background noise (with gel pieces, PCR product, etc.), and after consulting Kara and Mike, I decided to run the next round of SELEX with the solution acquired by the unstained gel, the one for which extraction appeared to be successful. -AW

Set up the bead-capture oligo complexes as before, did the initializing washes, and submerged the beads in the DNA solution, leaving it to incubate overnight (crossing fingers that there is sufficient DNA in the "DNA solution"

Today we move forward with round 2

Since at this point we are anxious to retain what DNA may have bound to the beads, we have decided to set a low threshold this round: we did a PABA wash after only 1 selection buffer wash. (On Friday we did 5 SB washes + an overnight wash.)

Even with such a low threshold, the Nanodrop gives us a reading of -1.4ng/mol (using sterile PABA soln as background)

Nothing to do but PCR amplify and hope for the best:

13 50uL reactions

25uL Q5

10uM F primer, 2.5uL

10uM R primer (w/ spacer), 2.5uL

20uL DNA

Split PCR product into 2 1.5mL tubes (300uL and 350uL)

Added 2.5-3 volumes (875uL) to each

Added 4uL (20ug) linear polyacrylamide

Centrifuged for 20min (before lab closed)

Would have liked to centrifuge longer, but lab was closing. Fortunately, this was long enough to produce a pellet.

Saved supernatant in case want to centrifuge again.

Added 100uL TE buffer to each

Saved at -20C

Next step is to run a gel to see whether we have DNA or not.

If we have DNA, we can move forward with gel extraction.

If we do not have DNA, we will return to our SELEX rd. 1 stock solution and progress from that point onward.