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Protocols

Protocols Archive

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.

General Mol. Biol. & Lab Topics

Useful External Links Collection    

Quick Links

Cycle Sequencing reaction recipes

 

DNA Sequencing

Qiagen Guide to Template Purification and DNA Sequencing

Cycle sequencing reaction recipes

PCR reaction  sequencing template preps

Plasmid sequencing template preps

Buffers for preparing and diluting cycle sequencing reactions

Thermal cycler programs for cycle sequencing

Cycle sequencing reaction clean-up methods
 

 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)

 

FragAnalysis

Sample loading recipes

Expt'l design strategies
to reduce costs and improve data quality

Fluorescent dyes and filter sets

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

 

Automated Sample Preparation

Qiagen kits and BioRobot Protocols
 

Real Time PCR

ABI SYBR Green mix

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
 

   

 

       
single channel pipetting 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
  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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PCR reaction sequencing template preparation methods

 

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 for 20 minutes.

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.

  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.
 

  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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Plasmid sequencing template preparation methods

  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.
  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
  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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Buffers for preparing and diluting cycle sequencing reactions

  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
 

  2.5x Dilution buffer for BDT v1.1 & 3.1

From the old 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
 

  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.
 

  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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Generic thermal cycler program for BigDye 1.1/3.1 cycle sequencing reactions

  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.
 
 

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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Cycle sequencing reaction clean up methods

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.

Multichannel pipetting 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.
 

The ever popular plate centrifuge 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
  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
       

Fragment Analysis

Sample preparation recipes for the ABI 3130XL

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
 

  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 3130XL.  Therefore the dilutions of the PCR products used on the 3730 will be different (greater dilutions) from those used on 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.

  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).
 
 

Useful experimental design strategies to reduce costs and improve data quality

  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.
 
  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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About fluorescent dye labels and 3100 "filter sets"

Who are JOE, NED, VIC, HEX, ROX, FAM, etc.?
 
Dye trade name      Rel. Ints'y

(6, 5)FAM Big Blue Dot

Dye chemical name.

Fluorescein, derivatized as NHS ester via a carboxyl at position 5 or 6.  
 

Abs.      Em.

495nm     520nm

  HEXMed Green Dot Hexachlorofluorescein, NHS ester.  Can only be used on 5' end of oligo.
 
535nm     555nm
  JOEMed Green Dot 6-carboxyl-4',5'-dichloro-2',7'- dimethoxyfluorecein, NHS ester
 
529nm     555nm
  ROXSmall Red Dot Carboxy X-rhodamine, NHS ester.
 
588nm     608nm
  TAMRASmall Yellow Dot Carboxy tetramethyl rhodamine, avail. as NHS ester, or direct linked.
 
559nm     583nm
  TETBig Green Dot Tetrachlorofluorescein.  NHS ester.  Can only be used on 5' end of oligo.
 
522nm     539nm
  NEDMed Yellow Dot ABI proprietary "yellow".
 
553nm     575nm
  VICBig Green Dot ABI proprietary "green".  Same emission wavelength as JOE, but narrower spectral peak and brighter signal.
 
538nm     554nm
  PETSmall Red Dot
 
ABI proprietary "red". 558nm     595nm
  LIZMed Orange Dot ABI proprietary "orange". 638nm     655nm
       
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.

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.
  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.
  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

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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Date last changed Sep 29th, 2016 @ 09:22:21 PDT