Latest revision as of 19:50, 19 October 2016

Stanford-Brown 2016

Aptamer Purification (DNAzyme, PQQ, SELEX) · Benchling

Aptamer Purification (DNAzyme, PQQ, SELEX)

Made with Benchling

Project: iGEM 2016

Authors: Michael Becich, Charles Gleason, Amy Weissenbach

Dates: 2016-08-15 to 2016-10-12

Monday, 8/15

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PQQ Aptamers Ordered (IDT):

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Aptamer 1: (15ADa9): /5Biosg/GA ACT AGA TCG CAG CCC ACC GGC GGA CAG CAT AGG ACG ACT GGC TCG AGC GCT CTA CTA CTG CGG CAT TTC TAC CCT GAT TTG TAG GAT CGA GGT AAT CC

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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

Tuesday, 8/30

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Made B&W Buffer

Wednesday, 8/31

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Made 1M Lysine-HCl Stock

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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.

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

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It will take at least a week to arrive.

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We still need to order the proper denaturing gel supplies.

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Designed PQQ Assay (Variables: 1:10 Dilutions, Real vs. Supplemental, Copper,Lysine, Aptamer)

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Jarrow - QH+PQQ - 60 Softgels purchased from VItamin Shoppe

Thursday, 9/1

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Made 2x Binding Buffer (100mM HEPES, 200mM KCl, 50mM MgCl₂, 10mM CaCl₂) for incubating PQQ with aptamer

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Made 1M CuSO₄ stock

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Washed Magnetic Streptavidin Dynabeads

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

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Immobilized Biotinylated Oligonucleotides on beads (30 min incubation)

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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

Friday, 9/2

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

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

A

B

C

D

E

F

G

H

I

J

K

L

M

1

A H2O2 Standard

STD1

STD2

STD3

STD4

STD5

STD6

STD7

STD8

STD9

STD10

STD11

STD12

2

2.5

1.25

0.625

0.3125

0.15625

0.073125

0.036

0.018

0.009

3

B Pure PQQ, Aptamer 1

SPL1

SPL5

SPL9

SPL13

SPL17

BLK

SPL25

SPL29

SPL33

SPL37

SPL41

BLK

4

5

C Supp PQQ, Aptamer 1

SPL2

SPL6

SPL10

SPL14

SPL18

BLK

SPL26

SPL30

SPL34

SPL38

SPL42

BLK

6

7

D Pure PQQ, Aptamer 2

SPL3

SPL7

SPL11

SPL15

SPL19

BLK

SPL27

SPL31

SPL35

SPL39

SPL43

BLK

8

9

E Supp PQQ, Aptamer 2

SPL4

SPL8

SPL12

SPL16

SPL20

BLK

SPL28

SPL32

SPL36

SPL40

SPL44

BLK

10

11

Racemic Lysine

No Racemic Lysine (Control with racemic lysine)

Peptidal Lysine

No peptidal Lysine (Control with peptidal lysine)

12

Table1

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

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

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

IMG_3854.JPG

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

IMG_4574[1].JPG

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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:

PQQ_Experimentv1.xlsx

Thursday, 9/8

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

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Ordered correct denaturing PAGE gel, BIORAD Mini-PROTEAN 10% TBE-Urea Precast Gels

Friday, 9/9

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Washed bead complexes + DNA, washed with pABA

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

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

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Used remainder of flow-through for PCR amplification!

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Thermocycler settings:

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98C (30s)

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[98C (10s)

50C (20s)

72C (20s)] x30

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72C (2min)

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4C (infinity)

Monday, 9/12

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Pooled and precipitated DNA with ethanol in presence of linear polyacrylamide

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

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

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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.

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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.

Wednesday, 9/14

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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.

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

Thursday, 9/15

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

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

IMG_3667.JPG

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

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

Monday, 9/19

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

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

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Followed protocol to make ssDNA ladder:

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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)

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Incubate @ 70C for 5min

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Electrophorese sample in denaturing gel, 2ug/lane

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

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

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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

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

Tuesday, 9/20

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

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

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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.)

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

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

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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.

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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.)

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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.

Thursday, 9/22

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

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

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Very difficult to tell whether or not this step worked.

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(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.)

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

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16 tubes

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300uL sodium acetate soln

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750uL ethanol

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3.5uL LP

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Centrifuged at max (13.3rpm) for 45min

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

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

Friday, 10/7

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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

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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"

Saturday, 10/8

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Today we move forward with round 2

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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.)

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

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

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13 50uL reactions

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25uL Q5

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10uM F primer, 2.5uL

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

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20uL DNA

Wednesday, 10/12

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

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Added 2.5-3 volumes (875uL) to each

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Added 4uL (20ug) linear polyacrylamide

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Centrifuged for 20min (before lab closed)

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

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Saved supernatant in case want to centrifuge again.

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Added 100uL TE buffer to each

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Saved at -20C

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

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

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