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Genotyping - faster and better; part 2Dear ,
before we will have a nice and relaxed X-mas break, we would like to inform you about new customer references from Belgium, Finland; Spain and Japan. We would like to say thank you very much, especially to Matti from Finland, Javier from Spain and Aki from Japan because we know it's not easy to get these data from customers - GREAT JOB!!!
All data shown here (and more) are available on our WEB page. Furthermore you'll find a section FAQ and customer references for DNAreleasy.
All customers mentioned here, have used succesfully Kapa enzymes and/or our DNAreleasy kit. You can use these references as well to convince customers - Have fun and success
1) DNAreleasy and Kapa 2G Robust HS - a powerful combination for carcinoma cell culture
2) Kapa Blood PCR kit for the detection of the Thiopurin S-methyl-transferase gene
3) Food Control: Detection of Salmonella, Streptococcus suis and other strains by using Kapa 2G Fast
4) Dog genotyping - the recommended enzyme is Kapa 2G Fast HS
1) DNAreleasy and Kapa 2G Robust HS - a powerful combination for carcinoma cell culture
Direct PCR with HT-29 colon carcinoma cells and DNAreleasy
Customer: University of Turku, Faculty of Medicine, Pathology; Ms. Gudrun Wahlström
Statement: This kit worked very well without any optimization. It is a very simple procedure and the combination with the Kapa 2G Robust HS enzyme leads to superior results.
| 1 |
DNA marker |
| 2 | DNAreleasy lysate, cell culture of HT-29 colon carcinoma cells, amplified with Kapa 2G Robust HS |
| 3 |
Plasmid DNA, amplified with Kapa 2G Robust HS |
| 4 |
Plasmid DNA and cell culture lysate, amplified by Kapa 2G Robust HS |
| 5 |
Negative control |
| 6 |
DNAreleasy lysate, cell culture of HT-29 colon carcinoma cells, amplified with Kapa 2G Robust HS (a different primer set was used) |
| 7 |
Plasmid DNA, amplified with Kapa 2G Robust HS (a different primer set was used) |
| 8 |
Plasmid DNA and cell culture lysate, amplified by Kapa 2G Robust HS (a different primer set was used) |
| 9 |
Negative control |
2) Kapa Blood PCR kit for the detection of the Thiopurin S-methyl-transferase gene
Lane 1-6: Kapa PCR Blood kit Buffer A; Lane 7-12 Kapa PCR Blood Kit buffer B
Customer:
Dr. Luis López-Fernández; Laboratory of Pharmacogenetics and Pharmacogenomics, Department of Pharmacy, Hospital General Universitario Gregorio Marañón, Madrid. Spain
Application:
PCR experiment was performed for the discrimination of different alleles of the Thiopurin S-methyl-transferase gene. Both reactions contains a mix of specific primer set for the Wild Type or mutant sequence and a primer pair as positive control. The amplification is as described by Roberts et al. (2004) in Clinica Chimica Acta 341:49-53 (read more) using the Kapa Blood PCR kit (Kapa Biosystems) and the following protocol:
Protocol:
15min 95°C, 40 cycles (30sec 94°C, 90sec 60°C, 90sec 72°C), 5min 72°C.
Finally, 1 µL of each reaction was analyzed in a Bioanalyzer 2100 (Agilent) with the DNA1000 kit (Agilent).
Conclusion:
The are two different PCRs and the expected band sizes were 200, 340 and 600bp for the first PCR and 330 and 600bp for the second.The gel shows 2 different buffer systems (lane 1-6 Buffer A; lane 7-12 buffer B), included in the Kapa PCR Blood kit. Best results were obtained with buffer B (lanes 6-12). By using this kit it is not anymore necessary to purify the genomic DNA from whole blood asa template preparation step for PCR.
Customer: Inspection Centre , Japan
Introduction:
Detection of SalmonellaTyphimurium (ST) and Salmonella Enteriditis (SE); Streptococcus suis and Erysipelothrix rhusiopathiae by using colony PCR and a fast PCR enzyme; (Kapa2G Fast PCR Enzyme from Kapa Biosystems). Efficiency and time was measured in comparison to a regular TAQ
Methods:
PCR was performed as follows.
| Bacterial species | Annealing temperature | References | Amplification products | |
|
Salmonella Typhimurium(ST) Enteriditis(SE) |
58℃ |
JOURNALOF CLINICAL MICROBIOLOGY |
Aug 2001 ,p.2981-2983 |
Typhimurium:1000bp Enteriditis:250bp |
| Streptococcus suis |
52℃ |
JOURNALOF CLINICAL MICROBIOLOGY | July 2004 ,p.3169-3175 | 294bp |
| Erysipelothrix rhusiopathiae |
54℃ |
JOURNALOF CLINICAL MICROBIOLOGY | June 1994 ,p.1526-1531 | 407bp |
The PCR reaction conditions and reaction settings are as kit protocol. Kapa2G Buffer A was used. Colony PCR was performed, the amount of bacteria of one platinum loop was boiled. After a quick spin different volumes of the supernatant were applied to PCR.
Cycle conditions (for both enzymes):
95° = 1 minute
Denaturation = 95° = 10 seconds; Annealing = 10 seconds; Extension = 72°C = 5 seconds
35 cycles; last step: 72°C = 30 seconds; total time 1 hour 9 minutes.
PCR reactions were the same for Kapa2G Fast and the regular Taq.
Detection of SalmonellaTyphimurium (ST) and Salmonella Enteriditis (SE) < 1st and 2nd test>
5µl of each PCR reaction was loaded on the agarose gel
| lane 1 | DNA marker |
| lane 2 | SalmonellaTyphimurium (ST) |
| lane 3 | Salmonella Enteriditis (SE) |
| lane 4 | SalmonellaTyphimurium (ST) |
| lane 5 | Salmonella Enteriditis (SE) |
| lane 6 | Negative control |
| lane 7 | Salmonella Typhimurium (ST) |
| lane 8 | Salmonella Enteriditis (SE) |
| lane 9 | Salmonella Typhimurium (ST) |
| lane 10 | Salmonella Enteriditis (SE) |
| lane 11 | Negative control |
3rd test
 |
lane 1 | 100 bp ladder |
| lane 2 | ST (1000 bp) Kapa 2G Fast | |
| lane 3 | SE (250 bp) Kapa 2G Fast | |
| lane 4 | negative control Kapa 2G Fast | |
| lane 5 | ST (1000 bp) regular TAQ | |
| lane 6 | SE (250 bp) regular TAQ | |
| lane 7 | negative control regular TAQ |
Detection of Streptococcus suis and Erysipelothrix rhusiopathiae by using Kapa 2G Fast
 |
lane 1 | 100 bp ladder |
| lane 2 | Erysipelothrix rhusiopathiae sample 1 (1st test) |
|
| lane 3 | Erysipelothrix rhusiopathiae sample 2 (2nd test) |
|
| lane 4 | Erysipelothrix rhusiopathiae sample 3 (3rd test) |
|
| lane 5 | Erysipelothrix rhusiopathiae, mixed pig streptococcus | |
| lane 6 | Swine streptococcic sample 1 (1st test) | |
| lane 7 | Swine streptococcic sample 2 (2nd test) | |
| lane 8 | Swine streptococcic sample 3 (3rd test) |
Conclusions:
The regular TaqDNA polymerase failed sometimes and even if a PCR product was found, intensity of the Kapa 2G Fast enzyme was better.
Both difficult to amplify strains Streptococcus suis and Erysipelothrix rhusiopathiae has given good and consitent amplification by using KAPA2GFast kit.
Another aspect was to reduce the time of the PCR process and this is possible by using the Kapa 2G Fast enzyme:
| Reaction time with the regular TAQ | Reaction time and with the fast enzyme KAPA 2G Fast | |
| Salmonella | 2h; 57 min. | 1h; 09 min. |
| Streptococcus suis | 3h; 15 min. | 1h; 09 min. |
| Erysipelothrix rhusiopathiae | 2h; 20 min. | 1h;09 min. |
4) Dog genotyping - the recommended enzyme is Kapa 2G Fast HS
Dear Partner (from Ron McEwan, CEO of Kapa Biosystems)
Our distributor in
Apparently some Dog Breeders associations are now demanding the dogs are genotyped prior to breeding to help maintain the pedigree, so this might be an opportunity in other European countries, I have attached a statement from the Progenus website below.
“Progenus has been chosen by the Royal Saint Hubert Society for the pedigree control by DNA analysis. SRSH members MUST contact SRSH to obtain sampling kit”.
That's all for this year, I hope you like our newsletter and products. It is fantastic to work with you and I look forward to see a lot of you guys next year.
For now, we would like to wish you some relaxing days and a wonderful start into 2009. Keep the ball rolling
Very best regards
Juergen
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