General protocol
Kits codes and components are reagents KAPA Blood BCR Mix A and/or KAPA Blood BCR Mix B
Quick Notes
-KAPA Blood PCR Mixes (2x) contain all components required for Whole Blood PCR, except primers and template (blood).
-Use KAPA Blood PCR Mix A (2x) for assays employing sensitive fluorescent detection systems (e.g. for paternity testing using the Promega PowerPlex® 16 system).
-Use KAPA Blood PCR Mix B (2x) for GC rich amplicons, other difficult amplicons and assays based on analysis by agarose gel electrophoresis/ethidium bromide staining.
-Amplify DNA fragments directly from blood collected in EDTA anticoagulant tubes, on FTA® Elute Cards, Whatman 903® paper (“Guthrie cards” ) or regular filter paper.
-Not suitable for direct amplification from blood collected in anticoagulant tubes containing heparin.
-For amplification from human EDTA blood, use 1 - 20%v/v blood in the PCR.
-For GC-rich amplicons, include 5%DMSO in reactions.
-Spin PCR products to collect debris prior to analysis.
-Purification of PCR products with a standard PCR cleanup kit prior to RE digestion, DNA sequencing or dHPLC analysis is recommended.
1.Typical reaction setup using EDTA blood
| Reaction component | Final concentration | Per 50 μl rxn | Per 25 μl rxn | Per 10 μl rxn |
| PCR grade water | Up to 50.0 μl | Up to 25.0 μl | Up to 10.0 μl | |
| Forward primer (5 μM) | 0.25 μM | 2.50 μl | 1.25 μl | 0.50 μl |
| Reverse primer (5 μM) | 0.25 μM | 2.50 μl | 1.25 μl | 0.50 μl |
| DMSO (100%) or Tween 20® (2%) | (5%or 0.1%v/v) | (2.50 μl) | (1.25 μl) | (0.50 μl) |
| KAPA Blood PCR Mix A or B (2x) | 1x | 25.0 μl | 12.5 μl | 5.00 μl |
| EDTA blood | 10%v/v | 5.0 μl | 2.50 μl | 1.0 μl |
2. Cycling parameters
| Cycling step | Time&temperature |
| Initial denaturation | 5 min at 95ºC |
| Denaturation | 30 sec at 95ºC |
| Annealing | 30 sec at optimal Ta |
| Extension | 1 min/kb at 72ºC |
| No. of cycles | 30 - 40 |
| Final extension | 0 - 1 min/kb at 72ºC |
3. Products
| Description |
Size |
Catalog# |
| KAPA Blood Readymix PCR Kit A | 500 rxn | KK7002 |
| 1 000 rxn | KK7004 | |
| KAPA Blood Readymix PCR Kit B |
500 rxn | KK7003 |
| 1 000 rxn | KK7005 |
4. Results
Amplification of a 459 bp fragment of exon 19 of the human Duchenne muscular dystrophy gene directly from whole human blood, using KAPA Blood PCR Mix B.
Reactions (50 µL) contained different amounts of blood (0 - 20%v/v, as indicated) from EDTA anticoagulant tubes stored at 4°C (lanes 1 - 6), or punches from an FTA® Elute Card (lane 7) or a “Guthrie card” (lane 8) as template. Control reactions, performed with 10 ng or 1 ng purified human genomic DNA as template and KAPA Blood PCR Mix B (lanes 9 and 10), or wild-type Taq polymerase (lanes 11 and 12) are included on the right. A standard 3-step cycling profile (35 cycles) with an initial denaturation of 5 min (95°C) and 1 min extension time per cycle was used in all reactions.