KS USDA, St. AmandRevised on 13-April-2015

KASP Protocolusing the ABI7900

The purpose of this protocol is to explain how to perform KASP assays, including how to set up the assays and how to analyze them afterwards. KASP reaction mix contains all the components necessary for performing PCR, except for 1) the appropriate KASP primers, 2) the sample DNA, and 3) additional MgCl2 if needed. For KASP assays you will need to use an optically clear plate, not the ones we usually use for PCR and ABI runs. We currently use the Thermo-Scientific AB-1384 plates (no black lettering on top of plates).NOTE: the KASP reaction mix is light sensitive and care must be taken to minimize the amount and total time that light falls on the reactionmix or on any tubes or plates containing KASP reactionmix.Also, you must include at lease 1 sample that has NO DNA (water blank, NTC) as a control. As usual, you should always include known positive and negative control DNA samples.

Primers and Assay Reaction Setup

  1. Defrost frozen DNA samples and primers in the water bath (65°C), 15 min for DNA plates and 2min maximum for primers and the KASP reaction mix. Vortex the DNA plates and primers briefly then centrifuge them for 1 min at max rpm. Hold at 4℃ or on ice until needed.
  2. If you are using our lab-prepared KASP primer pools, then skip to step 4 below.
  3. If you are assembling your own KASP primer pool from new, dry, primers, resuspend your primers in TRIS-HCl (pH 8.0 to 8.5) and dilute them to 100 uM. Combine your 2 forward primers with the common reverse primer as shown in the table below. You will then need to enter your KASP marker data into the SDS software using the "Marker Manager" and "Detector Manager".

ul / Stock / FC in assay mix
120 / AL1 100pm/ul (100 uM) / 12 / pm/ul
120 / AL2 100pm/ul (100 uM) / 12 / pm/ul
300 / C1 100pm/ul (100 uM) / 30 / pm/ul
460 / TrisHCl (10mM, pH8.3)
1000 / Total vol. of assay mix
  1. Prepare the master mix on ice and in the dark as much as possible, by adding water, additional MgCl2, primer pool, and then the KASP reaction mix, in that order.The KASP reaction mix, when used at 1X, has 2.5 mM MgCl2. Most KASP assays work well without additional MgCl2, but additional MgCl2 often increases the yield of the assay and drives the clusters further apart. The KASP reaction mix is very expensive. Waste must be kept to a minimum. Use only a 4 to 6 ul total reaction volume. Use a minumum of 40ng of DNA and 200ng is better.
  2. Example master mix for a 4 ul total volume with no extra MgCl2:

Single Rxn Vol / 4 / ul
Number of Rxns / 100 / Rxns
Components in / Stock / Single Rxn / Final / Total
Master Mix / conc. / Units / Vol (ul) / conc. / Units / Vol (ul)
Nanopure Water / 0.0000 / 0.00 / ul
2x KASP reaction mix / 2.00 / X / 2.0000 / 1.0000 / X / 200.00 / ul
72x primer assay mix / 72.00 / X / 0.0556 / 1.0000 / X / 5.56 / ul
MgCl2 (additional) / 50.00 / mM / 0.0000 / 2.5000 / mM / 0.00 / ul
1.944 / DNA, ul
2.0556 / MM/Rxn, ul / 205.56 / ul
  1. Example master mix for a 4 ul total volume with extra MgCl2:

Single Rxn Vol / 4 / ul
Number of Rxns / 100 / Rxns
Components in / Stock / Single Rxn / Final / Total
Master Mix / conc. / Units / Vol (ul) / conc. / Units / Vol (ul)
Nanopure Water / 0.0000 / 0.00 / ul
2x KASP reaction mix / 2.00 / X / 2.0000 / 1.0000 / X / 200.00 / ul
72x primer assay mix / 72.00 / X / 0.0556 / 1.0000 / X / 5.56 / ul
MgCl2 (additional) / 50.00 / mM / 0.0400 / 3.0000 / mM / 4.00 / ul
1.904 / DNA, ul
2.0956 / MM/Rxn, ul / 209.56 / ul

Once you have made your KASP mix, dispense it into the optically clear plate. Cover the mix and the plate with cardboard file covers. Only remove the cover to go into the mix and dispense. KEEP THE KASP MIX IN THE DARK AS MUCH AS POSSIBLE. Make certain you dispense the appropriate mix into the appropriate quadrants, and use a multichannel pipetter for more rapid dispensing.

Plate Record File

  1. Before you cycle (run) the PCR reaction, you must perform a pre-read analysis. IF YOU FORGET THIS STEP, YOU WILL HAVE TO START OVER!
  2. To perform apre-read analysis, first create a text-only plate record file (setup file). There is an example file on Dolly in the sequencing folder. Example using 1 marker:

*** SDS Setup File Version3

*** Output Plate Size384

*** Output Plate IDRhtD1-KASP-Example-Run-1-Marker

*** Number of Detectors2

DetectorReporterQuencherDescriptionCommentsAIF Assay ID

A, Rht2-D1b-Short, HEXVICRhtD1-KASP

C, Rht2-D1a-Tall, FAMFAMRhtD1-KASP

*** Number of Markers1

MarkerAlleleXAlleleYDescriptionCommentsAlleleXBaseAlleleYBase

RhtD1-KASPC, Rht2-D1a-Tall, FAMA, Rht2-D1b-Short, HEXCA

WellSample NameDetectorTask

1Quad-1,96-A01,384-A01,7900-1C, Rht2-D1a-Tall, FAM/A, Rht2-D1b-Short, HEXUNKN

49Quad-1,96-B01,384-C01,7900-49C, Rht2-D1a-Tall, FAM/A, Rht2-D1b-Short, HEXUNKN

97Quad-1,96-C01,384-E01,7900-97C, Rht2-D1a-Tall, FAM/A, Rht2-D1b-Short, HEXUNKN

145Quad-1,96-D01,384-G01,7900-145C, Rht2-D1a-Tall, FAM/A, Rht2-D1b-Short, HEXUNKN

193Quad-1,96-E01,384-I01,7900-193C, Rht2-D1a-Tall, FAM/A, Rht2-D1b-Short, HEXUNKN

...

384NTC-Quad-4,96-H12,384-P24,7900-384C, Rht2-D1a-Tall, FAM/A, Rht2-D1b-Short, HEXNTC

WellSample NameMarkerTask

1Quad-1,96-A01,384-A01,7900-1RhtD1-KASPUNKN

49Quad-1,96-B01,384-C01,7900-49RhtD1-KASPUNKN

97Quad-1,96-C01,384-E01,7900-97RhtD1-KASPUNKN

145Quad-1,96-D01,384-G01,7900-145RhtD1-KASPUNKN

193Quad-1,96-E01,384-I01,7900-193RhtD1-KASPUNKN

...

384NTC-Quad-4,96-H12,384-P24,7900-384RhtD1-KASPNTC

Plate Pre-Read

  1. Before you cycle (run) the PCR reaction, you must perform a pre-read analysis. IF YOU FORGET THIS STEP, YOU WILL HAVE TO START OVER!
  2. To perform a pre-read analysis, first turn on the power to Toto. This allows the laser to warm up.
  3. Make sure the plate is covered with optically clear film and not a rubber mat. The plate MUST have been centrifuged to get rid of all the air bubbles; otherwise, the pre-read will be invalid.
  4. Move the plate record from Dolly to the computer desktop on Toto.
  5. Open the SDS software (SDS 2.4).
  6. Go to File, then New Plate Wizard, then AD (allelic discrimination), then Next. Select 384 well clear plate and Setup File.
  7. Browse to get your plate record and copy the name.
  8. Click Open and click Finish.
  9. Go to File, then Save As, then paste the name from the plate record. Save it to the Desktop.
  10. Click on the Instrument tab to open this window. Click Connect and wait for Toto to respond, then click Open/Close. Do not block the door. While the door is opening, take a Kimwipe and wipe the top of the plate.
  11. Check the plate for broken wells. If your plate has broken wells, DO NOT PUT IT IN THE INSTRUMENT! Doing so will cause contamination in the block. If there are broken wells, you must transfer your samples to a new plate before performing the pre-read.
  12. Put the plate in the plate holder with the A1 well on the A1 corner, then hit Pre-Read. The door will close automatically.
  13. Once the instrument reads “Run completed successfully” click OK.
  14. Hit Open/Close and remove your plate, then hit Open/Close again to close the instrument door. Do not leave the instrument door open.
  15. Click Disconnect and close the instrument software.
  16. Turn off the instrument and make backup copies of the files to your folder, plus copy them to Dolly. Do NOT leave the instrument on. Do NOT leave your files on the desktop.

Plate Post-Read and Data Scoring

  1. Put your plate on any thermocycler and run the appropriate program. There are 3 different KASP programs (KASP, KASPXC, and KASP60) so make certain you use the correct one. The KASP programs only take ~ 2 h to complete, as they are relatively fast.
  2. After the KASP PCR program is complete, centrifuge your plate at 5,700 RPM and then turn Toto on. On Toto, double click on your SDS version of the file. Go to the Instrument tab and hit Connect, then Open/Close. Clean your plate top with a Kimwipe, then click Post Read. The program should automatically save the file. Check the Instrument tab to see if there is a date/time stamp on the post-read. If not, be certain you save the file.
  3. Once the read is done, click the green Run arrow (►). A warning screen may come up; if it does, click OK.
  4. Go to the Results tab. Grab the divider and make the chart image square.
  5. The little black squares on the imageare the non-template controls. They should be near the 0:0 origin. If not, then they have some DNA contamination in them.
  6. If your assay worked well and the clusters are widely separated, then the samples should be auto-scored. If not, they will each be grey  marks.
  7. Use the lasso tool to encircle the  marks on the "Y" axis. Use the drop-down menu on the Call tab to manually score these as the "Y" allele. Do the same for the "X" axis samples and score them as the "X" allele. Confirm that your controls are in the correct allele cluster.
  8. Samples in the middle cluster, if present, are heterozygous or heterogeneous.
  9. Once all samples are scored, look at the plate layout and check for 's. If there are any, select and score them.
  10. You can right-click on the image and save the chart as a .jpg file.
  11. To save the genotype calls, go to File, then Export, then Results Table, all wells, and latest format, and save the file to the Desktop.
  12. Do NOT leave your plate in the instrument. Go to the Instrument tab, then Open/Close, and remove your plate. Hit Open/Close again and then Disconnect. Close the program and all associated windows.
  13. Do NOT leave your fileson the Desktop; they WILL be deleted. Put them on Dolly and move them into to your own folder.
  14. Turn off the instrument. Leaving it on greatly shortens the laser life.