Team:Stanford-Brown/SB16 Notebooks AptamerPurification


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
PQQ Aptamers Ordered (IDT):
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
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
Made B&W Buffer
Wednesday, 8/31
Made 1M Lysine-HCl Stock
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.
Designed PQQ Assay (Variables: 1:10 Dilutions, Real vs. Supplemental, Copper,Lysine, Aptamer)
Jarrow - QH+PQQ - 60 Softgels purchased from VItamin Shoppe
Thursday, 9/1
Made 2x Binding Buffer (100mM HEPES, 200mM KCl, 50mM MgCl2, 10mM CaCl2) for incubating PQQ with aptamer
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
Friday, 9/2
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
A
B
C
D
E
F
G
H
I
J
K
L
M
1
A H2O2 StandardSTD1STD2STD3STD4STD5STD6STD7STD8STD9STD10STD11STD12
2
2.51.250.6250.31250.156250.0731250.0360.0180.009
3
B Pure PQQ, Aptamer 1SPL1SPL5SPL9SPL13SPL17BLKSPL25SPL29SPL33SPL37SPL41BLK
4
5
C Supp PQQ, Aptamer 1SPL2SPL6SPL10SPL14SPL18BLKSPL26SPL30SPL34SPL38SPL42BLK
6
7
D Pure PQQ, Aptamer 2SPL3SPL7SPL11SPL15SPL19BLKSPL27SPL31SPL35SPL39SPL43BLK
8
9
E Supp PQQ, Aptamer 2SPL4SPL8SPL12SPL16SPL20BLKSPL28SPL32SPL36SPL40SPL44BLK
10
11
Racemic LysineRacemic LysineRacemic LysineRacemic LysineRacemic LysineNo Racemic Lysine (Control with racemic lysine)Peptidal LysinePeptidal LysinePeptidal LysinePeptidal LysinePeptidal LysineNo peptidal Lysine (Control with peptidal lysine)
12
Table1
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.
IMG_3854.JPG
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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
Made Selection Buffer, TE Buffer, TBE Buffer, more B&W Buffer
Ordered correct denaturing PAGE gel, BIORAD Mini-PROTEAN 10% TBE-Urea Precast Gels
Friday, 9/9
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)
Monday, 9/12
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.
Wednesday, 9/14
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.
Thursday, 9/15
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:
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.
We ordered proper materials for this gel, to avoid future complications: sampl loading dye, 10bp DNA ladder for denaturing gel
Monday, 9/19