Data Analysis & Protocols Archive
Protocols Archive |
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| A
collection of laboratory protocols and information which CGC users have
found to be particularly useful. The information is organized into
sections
on DNA sequencing, fragment analysis, automated sample preparation
and processing, and Real Time PCR. |
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DNA Sequencing |
Cycle Sequencing reaction
recipes |
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Quick
Links
DNA Sequencing |
ABI
BigDye 3.1 reactions from Biochemistry core Sequencing facility page |
General
recipes - 1/4 Reaction: 2 microliters RRM (ready reaction mix) 2 microliters 2.5x buffer 10 microliters total reaction volume 1/8 Reaction: 1 microliters RRM 3 microliters 2.5x buffer 10 microliters total reaction volume Please note that the dilution/sequencing buffer which ABI sells is 5x. It must be diluted 1:2 to make a working buffer of 2.5x to use in these recipes. The Biochemistry Sequencing Core Facility recommends a 1/4 reactions for most purposes: 2 uL of 2.5 x dilution buffer 2 uL of BigDye 3.1 RRM 4 pmoles of primer water + template + primer to 10 uL final volume. (If you need a 20 uL reaction increase the amount of buffer and water only) Recommended Template Quantity (for both 1/4 and 1/8 reactions) PCR product: 100 - 200 bp 25 ng 200 - 500 bp 50 ng 500 - 1000 bp 100 ng 1000 - 2000 bp 200 ng >2000 bp 400 ng Single-stranded DNA 300 +/- 100 ng Double-stranded Plasmid DNA 600 +/- 200 ng BACs, YACs, PACs 0.5 - 1.0 µg + 0.5 µl DMSO Genomic DNA 2.0 - 3.0 µg + 0.5 µl DMSO |
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| Automated
Sample Preparation Qiagen kits and BioRobot Protocols Real Time PCR ABI SYBR Green mix |
ABI BigDye 1/4 reaction with optimized Mg+2 concentration. From ABI tech. support, 4/2004 |
According to ABI tech support this recipe gives a better final magnesium concentration than other diluted BigDye reactions and works better on troublesome template samples. 2 uL BigDye 3.1 RRM 3 uL 5x buffer water + template + primer to 20 uL final volume (see below for primer and template quantities) |
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ABI BigDye full strength reaction. From the BigDye Terminator v3.1 Cycle Sequencing Kit Protocol book. |
8 uL BigDye 3.1 RRM 3.2 pmol primer template, as specified below Deionized water to 20 uL total reaction volume Template Quantity PCR product: 100 - 200 bp 1-3 ng 200 - 500 bp 3-10 ng 500 - 1000 bp 5-20 ng 1000 - 2000 bp 10-40 ng >2000 bp 20-50 ng Single-stranded DNA 25-50 ng Double-stranded Plasmid DNA 150-300 ng Cosmids, BACs, YACs, PACs 0.5 - 1.0 µg Genomic DNA 2.0 - 3.0 µg |
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Better Buffer 1/8 reaction for BigDye 3.1 From The Gel Company. Some users report improved results (stronger signals, more consistent success with difficult templates like PCR fragments and bisulfite-treated DNA) with Better Buffer as compared to ABI buffer or home-made Tris-magnesium buffers. |
5 uL Better Buffer 1 uL BigDye 3.1 RRM 2.4 pmole primer template + water to 15 uL final volume Recommended Template Quantities (preliminary values) PCR product 5-30 ng Plasmid 80-300 ng |
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| Amersham DYEnamic ET Terminator full strength reaction | 8 uL ET
Terminator RRM (ready reaction mix) 5 pmole primer 0.1-0.2 pmol template DNA water to 20 uL total reaction volume |
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Diluting ET Terminator reactions |
The sum of the ET RRM plus the dilution buffer must equal 40% of the reaction volume. Ex.: 2 uL RRM + 6 uL ET dilution buffer in a 20 uL reaction, or 2 uL RRM + 2 uL dilution buffer in a 10 uL reaction. Note: The ET Terminator reaction mix can also be substituted for the BigDye 3.1 RM in the Better Buffer reaction described above. |
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| Back to top |
PCR reaction sequencing template preparation methods |
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| PEG
precipitation Contributed by Dave Tank in the Olmstead Lab. This precipitation will remove most or all of the left over nucleotides and primers. |
Make up a stock solution of 20% PEG, 2.5M sodium chloride: 3 gm PEG 8000 2.19 gm NaCl bring to a total of 15 ml with good (distilled or MilliQ) water. (Editorial Note: Any solution that goes in to a precipitation should be free of particulates because they will end up in your pellet. Therefore, it is a good idea to filter solutions like this through a 0.45 or 0.2 micron syringe filter.) PEG precipitation in 96-well microplates 1. Add a volume of the PEG/NaCl solution to each well equal to the volume of the PCR reaction. In other words, add one volume of PEG/NaCl to each well. 2. Vortex the plate briefly and then place it in a 37°C water bath for 15 minutes. 3. Centrifuge at about 6000 x G, or maximum speed, in a swinging bucket plate centrifuge. 4. Invert the plate over the sink using a smooth circular motion to dump out as much of the supernatant as possible. Then blot the plate on a paper towel. 5. Place the plate upside down on a folded paper towel and centrifuge briefly at 600 x G to remove residual supernatant. Briefly means you use "Hold" for the time setting and just allow the centrifuge to reach 600 x G and turn it off. 6. Add 125 uL of cold 70% ethanol to each well, invert the plate using a smooth circular motion to dump as much ethanol as possible and then blot the plate on a paper towel. 7. Place the plate upside down on a folded paper towel and centrifuge briefly at 600 x G to remove residual supernatant. 8. Resuspend the samples in an appropriate amount of dH2O. |
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| ExoI/SAP
treatment Thanks to Christine Pince in the Bradshaw lab. ExoI digests single stranded DNA and thus eliminates the primers. Shrimp alkaline phosphatase removes the phosphates from the leftover dNTPs, inactivating them. |
For each 10uL PCR rxn add: 1.5 units SAP (shrimp alkaline phosphatase), typically 1unit/uL 10 units of ExoI (exonucleaseI), typically 20unit/uL Incubate at 37°C for 30 minutes Then 80°C for 15 minutes to deactivate the enzymes. Use the ExoSAP treated product as sequencing template within a few days. There is some residual nuclease activity present that will degrade the sample over time. Important note on ethanol precipitations of sequencing reactions - Experience has shown that ethanol precipitations of cycle sequencing reactions done using ExoSAP treated templates should use a recipe that contains EDTA. This prevents large dye blobs from occurring in the first 150 bases of the sequence reads. |
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| Other
methods and kits. |
There are a number of PCR clean up kits available. - Qiagen QiaQuick 96 kit, a 96-well format kit which can be run on the BioRobot or by hand with a centrifuge or vacuum manifold. - Montage ultrafiltration kit from Millipore. This could be done on the BioRobot by modifying the Montage sequence reaction clean up program slightly. - ExoSAPit kit from Amersham, which is sold at Biochem stores. - Spin columns from various manufacturers. |
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| Back to top |
Plasmid sequencing template preparation methods |
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| Qiagen
kits The Qiagen plasmid minipreps, produced by the QiaPrep and QiaPrep Turbo kits, are considered by many to be the "gold standard" of quality in plasmid preps. |
The Qiagen kits are avaliable in sizes from single preps to 96-well plates. They all use an alakaline lysis of he bacterial culture followed by binding the plasmid DNA to a silica filter. The bound DNA is washed with a special ethanol-containing buffer, dried, and then eluted in a low-salt aqueous buffer. The CGC has a robotic protocol for the QiaPrep96 Turbo. The prep starts with 1 ml cultures grown in a 96-well deep well blocks. |
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| Eppendorf
kits Eppendorf has a line of plasmid prep kits called PerfectPrep Vac Direct Bind kits. They work on the same principle as the Qiagen kits. |
The Eppendorf kits use the same principle of binding to silica membranes as the Qiagen kits use. They are generally less expensive than the Qiagen kits. The CGC has a robotic protocol for the Eppendorf PerfectPrep 96 plasmid kit. The prep starts with 1.2 ml cultures grown in 96-well deep well blocks |
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| Amersham
TempliPhi kit This kit allows one to make several micrograms of DNA directly from a bacterial colony pick without growing cultures or any purification process. |
This kit is based on the rolling circle replication process used by bacteriophage phi 29. The technique was described in a 2001 methods article from Genome Research Online. It has since been developed into the TempliPhi kit by Amersham. See how it works in this animation, reproduced with unofficial permission from an Amersham Biosciences promotional CD. It allows researchers to avoid the usual culturing and miniprep steps entirely. This makes sense both in terms of costs and time savings for users doing larger scale projects like ESTs. For smaller scale work the costs are about the same as doing mini-preps, according to Amersham. This quicktime animation illustrates the direct-from-colony process. |
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| Back to top | Buffers
for preparing and diluting cycle
sequencing reactions |
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Applied Biosystems, Inc. Proprietary buffer. |
5x Dilution Buffer for BDT v3.1 & 1.1 ABI: 1-800-874-9868, www.appliedbiosystems.com 1 ml cat # 4336697 $25 28 ml cat # 4336699 $740 233 ml cat # 4336701 $6120 |
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2.5x Dilution buffer for BDT v1.1 & 3.1 From the Biochem. Core Facility page. |
Tris HCl pH 9 - 175 mM MgCl2 - 1.25 mM Component Ordering Information: 1 M Tris HCl, pH 9 250 ml ~$20 Amresco 1-866-811-3697 1 M MgCl2 100 ml ~$30 Sigma 1-800-325-3010 |
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Better Buffer Proprietary buffer imported from England and distributed by The Gel Company. No one is saying what's in it. |
Better Buffer is packaged in 0.3 ml tubes for retail sale 10 tubes, DAF-10, $295.00 5 tubes, DAF-5, $170.00 The Gel Company 1-800-256-8596 Also sold at Biochemistry Stores. |
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Amersham ET Terminator dilution buffer |
DYEnamic ET Terminator Dilution Buffer Amersham: 800-526 3593, www1.amershambiosciences.com 1 mL cat. # US84002. $92.00 |
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| Back to top | Generic thermal
cycler program for BigDye
1.1/3.1 cycle sequencing reactions |
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From the BigDye Terminator v3.1 Cycle Sequencing Kit Protocol book. |
1. Rapid thermal ramp to 96 °C, hold 96 °C for 1 minute 2. Rapid thermal ramp to 96 °C, hold for 10 seconds 3. Rapid thermal ramp to 50 °C, hold for 5 seconds 4. Rapid thermal ramp to 60 °C, hold for 4 minutes 5. Back to step 2. 24 times (total of 25 cycles) 6. Rapid thermal ramp to 4 °C, hold for 5 minutes 7. End program. This will leave the block at room temperature until you are ready to purify the products. |
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| Generic thermal cycler program for ET Terminator cycle sequencing reactions | |||
From the DYEnamic ET Terminator Cycle Sequencing Kit instruction book. |
1. Rapid thermal ramp to 95 °C, hold for 20 seconds. 2. Rapid thermal ramp to 50 °C, hold for 15 seconds 3. Rapid thermal ramp to 60 °C, hold for 60 seconds 4. Back to step 1. 24 times (total of 25 cycles) 5. Rapid thermal ramp to 4 °C, hold for 5 minutes. 6. End program. This will leave the block at room temperature until you ready to purify the products |
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| Back to top | Cycle
sequencing reaction clean up methods |
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An important note about ethanol precipitations: Take care to resuspend the pellets in formamide or other loading buffer well. You can heat the samples in formamide briefly in the PCR machine, vortex them carefully, or a combination of the two. Generally, the less heating the better for sample stability. |
Simple sodium acetate/ethanol precipitation. Contributed by Ken Karol from the Olmstead group in Biology. The volumes quoted in this protocol are for 7 uL BigDye reactions. You will have to adjust volumes proportionally for other reaction volumes. |
ABI recommendations for ethanol precipitation of cycle sequencing products are to achieve a final concentration of 59% ethanol and 90 mM NaOAc, pH 4.8. This reduces the amount of unincorporated flourescent terminators in the precipitated DNA, while minimizing loss of small fragments and maximizing overall signal strength. Recipe assumes 7 uL sequencing reactions in a 96-well plate. 1. Make a premix of: 60 uL 3M NaOAc, pH 5.6 290 uL H20 1250 uL 100% EtOH 2. To each 7 uL sequencing reaction add 14 uL of the premix. 3. Vortex carefully to avoid splashing contents of wells into each other. 4. Hold at room temperature for 15 minutes. 5. Spin 20-30 minutes at full speed (5760 x G in our SIGMA plate centrifuge). 6. Invert the plate to drain it and place it upside down on a folded paper towel. 7. Add 125 uL of 70% EtOH to each well, mix by gently inverting the covered plate. Re-use the sealing film from the cycle sequence reaction as the cover. 8. Spin the plate at full speed for 10 minutes (right side up!) 9. Pour off the 70% ethanol and then briefly spin the plate inverted on top of a folded paper towel: Use the Hold setting for spin time. Let the speed just reach 600 x G, then stop the centrifuge. If you aren't going to load the reactions in the sequencer right away, store the reactions as dry pellets in the freezer. Resuspend the samples in formamide immediately before loading them in the sequencer. |
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Amersham DYEnamic ET Terminator ethanol precipitation. |
Amersham's recommendations for ethanol precipitation are different from ABI's. Amersham scientists have found that including EDTA in ethanol precipitations dramatically reduces the amount of unincorporated fluorescent terminators in the product pellets. Amersham cycle sequencing kits come with an ethanol precipitation buffer consisting of 1.5M NaOAc, 250 mM EDTA, pH > 8.0. Recipe assumes 20 uL sequencing reactions in a 96-well plate. 1. Add 2 uL (0.1 volumes) of NaOAc/EDTA buffer to each 20 uL reaction. 2. Add 80 uL of 95% ethanol to each well and mix using a vortex mixer. 3. Centrifuge at room temperature for 20-30 minutes at full speed. 4. Place the plate upside down on a folded paper towel and centrifuge for 1 minute at 300 x G to remove supernatant. 5. Add 100 uL 70% EtOH to each well, mix gently 6. Spin the plate at full speed for 10 minutes (right side up!) 7. Pour off the 70% ethanol and then briefly spin the plate inverted on top of a folded paper towel for less than a minute: Use the Hold setting for spin time. Let the speed just reach 600 x G, then stop the centrifuge. |
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Spin columns. Faster but more expensive. (You can save $$$ by loading your own spin columns or 96-well filter plates with G-50.) |
Use spin columns or 96-well plate format spin column arrays like the Amersham Autoseq G50 spin columns sold at Biochemistry Stores, or the Edge Biosystems 96-well gel filtration spin plates. http://www.edgebio.com/products/ProductList.php?pg=1 |
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Ultrafiltration. Not quite as fast as spin columns, not quite as expensive. Can be automated on the BioRobot. |
Use the Montage 96-well ultrafiltration plate kit from Millipore. This process uses vacuum instead of centrifugation, so it can be automated on the BioRobot. http://www.millipore.com/catalogue.nsf/docs/C7484 |
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Fragment Analysis |
Sample
preparation recipes for the ABI 3100 |
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| Back to top | Fragment analysis sample
preparation. From the Bradshaw Lab, 5/04 |
This
recipe assumes the PCR reaction has been carried out in 10 uL total
volume to keep costs down. Mix 1 uL of PCR reaction with 9 uL of water to make a 1:10 dilution. This would be done in a 96-well plate for a high throughput experiment. The necessary degree of dilution will vary for different PCR products (some trial and error required). If multiplexing, mix together equal amounts of 1:10 dilutions of the various PCR products to be multiplexed. This would be done in a different row, or a different plate if you are working on a large scale experiment. To prepare samples for loading into the 3100: 1 uL of diluted sample or multiplex mixture 0.5 uL of size standard 15.5 uL of formamide |
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| Alterations
to the fragment
analysis sample recipe for the ABI
3730. |
The ABI
3730 has more sensitive optical detection of the fluorescence emitted
by the labelled primers than the 3100. Therefore the dilutions of
the PCR products used on teh 3730 will be different from those used ont
the 3100/3130xl. You will also want to use less of the ROX
size standard in each run. We have found that using 5uL of HD400 ROX for each run of 48 samples provides a nice peak height in the 3730 and saves the user some money. |
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General Comment: |
You should expect to experiment a bit to determine the best degree of dilution of your fluorescent labeled PCR products in order to produce peaks in the same intensity range as the size standards on the 3100. The optimal intensity for the DataCollection software is peak values in the range of 1000 - 4000 rfu's (relative fluorescence units). |
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| Useful
experimental design strategies to reduce costs and improve data quality |
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Cost-saving primer design |
It is possible to make just one generic M13 fluorescent labeled primer for each color of dye and then use these with any number of specific unlabeled PCR primers. NATURE BIOTECHNOLOGY, Vol. 18, FEB. 2000. |
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| Dealing with +A bands. |
Taq DNA
Polymerase often adds an A to the end of a PCR product. This can
confuse data analysis, especially when it occurs at less than 100%
efficiency and in combination with stutter in a repeated sequence like
a microsatellite. ABI has a user
bulletin which discusses some strategies for either driving
addition of the A or preventing it. |
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| Back to top | About
fluorescent dye labels and 3100 "filter sets" |
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Who are JOE, NED, VIC, HEX, ROX, FAM, etc.? |
Dye trade name Rel. Ints'y (6, 5)FAM  |
Dye chemical name. Fluorescein, derivatized as NHS ester via a carboxyl at position 5 or 6. |
Abs. Em. 495nm 520nm |
HEX |
Hexachlorofluorescein, NHS
ester. Can only be used on 5' end of oligo. |
535nm 555nm | |
| JOE |
6-carboxyl-4',5'-dichloro-2',7'-
dimethoxyfluorecein, NHS ester |
529nm 555nm | |
ROX |
Carboxy X-rhodamine, NHS ester. |
588nm 608nm | |
TAMRA |
Carboxy tetramethyl rhodamine,
avail. as NHS ester, or direct linked. |
559nm 583nm | |
TET |
Tetrachlorofluorescein.
NHS ester. Can only be used on 5' end of oligo. |
522nm 539nm | |
NED |
ABI proprietary "yellow". |
553nm 575nm | |
| VIC |
ABI proprietary "green".
Same
emission wavelength as JOE, but narrower spectral peak and brighter
signal. |
538nm 554nm | |
| PET |
ABI proprietary "red". |
558nm
595nm |
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LIZ |
ABI proprietary "orange". |
638nm
655nm |
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| 4 dye set for fragment analysis experiments on the 3100/3130xl/3730 | Filter set D, DS-30 matrix
standard. Installed in the CGC 3100's. |
6FAM (blue), HEX (green), NED
(yellow), ROX (red, reserved for size standards) |
Uses ROX-labeled size standards |
| 4 dye set which uses the
brighter VIC in place of HEX |
Filter set D, DS-31 matrix
standard. Not currently installed. |
6FAM (blue), VIC (green), NED (yellow), ROX (red, reserved for size standards) | Uses ROX-labeled size standards |
| 4 dye set which uses JOE in place of HEX | Filter set F, DS-32 matrix
standard. Not currently installed. |
6FAM (blue), JOE (green), NED (yellow), ROX (red, reserved for size standards) | Uses ROX-labeled size standards |
| 5 dye set available for fragment analysis experiments on the 3100/3130xl/3730 | Filter set G5, DS-33 matrix
standard. Not currently installed in the CGC. If demand develops..... |
6FAM (blue), VIC (green), NED (yellow), PET (red), LIZ (deep red, reserved for size standards, but displayed as orange) | Uses LIZ-labeled size standards |
Automated Sample Preparation |
Kits and
protocols currently available in the CGC for the BioRobot 8000. |
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| Back to top | QiaPrep 96 Turbo Qiagen |
High purity plasmid mini-prep. | A BioRobot kit, includes 4
QiaPlates to make 384 preps. |
| R.E.A.L. Prep 96 (rapid extraction alkaline lysis minipreps) Qiagen |
Fast plasmid prep, suitable
for PCR, works with plasmid, cosmid, and BAC DNA. |
A BioRobot kit, includes 4
Qiaplates to make 384 preps. |
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| QiaQuick PCR 96 Qiagen |
Quick PCR reaction clean up. |
A BioRobot kit, includes 4 Qiaplates to purify 384 PCR products. | |
| DNeasy 96 Tissue Qiagen |
Animal tissue genomic DNA
extraction |
Not a true BioRobot kit, so
extra volumes of some buffers may be needed for the Robot. |
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| DNeasy 96 Plant Qiagen This prep makes use of the MixerMill 300 96-sample format bead beater. |
Plant tissue genomic DNA
extraction |
Not a true BioRobot kit, so extra volumes of some buffers are needed for the Robot. | |
| PerfectPrep 96 Brinkmann/Eppendorf |
High purity plasmid mini-prep. Lower cost than the QiaPrep 96 Turbo kit. |
Not a true BioRobot kit, so extra volumes of some buffers are needed for the Robot. | |
Real Time PCR |
SYBR
Green reactions |
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| Back to top | ABI SYBR Green PCR Master Mix |
The
Master Mix is 2x. Just add your template and primer, and water to
bring the reaction to final volume. |
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