Ask the Experts: Ensuring a Successful PCR Every Time

Transcription

1 Ask the Experts: Ensuring a Successful PCR Every Time

2 Welcome Things to know before we get started

3 Housekeeping Application Widgets Movable and resizable Any issues with console Press F5 to refresh your browser Ready to answer your questions Use Q&A widget Poll questions Resource materials available Use Resource List widget Download resources Bookmark links

4 Technical Support Scientists For the Americas: Monday - Friday 7:00am-6:00pm CST techserv@promega.com

5 Technical Services Troubleshooting current experiment Figuring out alternative protocols for a difficult sample type Planning a new experiment, figuring out what products to use To learn more about a general science topic For understanding how a kit works and how it could be optimized To get customized web based training for a technique new to the lab

6 What are the components of PCR and their functions?

7 PCR The Basics

8 Components of a PCR reaction

9 PCR Cycling Parameters C Initial Denaturation Denaturation 2 minutes Denaturation seconds C Extension C 1-2 minutes Annealing seconds Final Extension 5-20 minutes 4-10 C Hold/Soak forever 1 Cycle Cycles 1 Cycle 1 Cycle

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16 All the components are needed to work together

17 Polymerase Options Thermus aquaticus - or Taq Brock & Freeze 1969 CREDIT: David Ward, Montana State University

18 What is Hot Start?

19 Taq Hot Start Hot Start MM Taq Hot Start Hot Start MM Hot Start

20 Why would you use a Master Mix?

21 Custom PCR Protocols Tool to Make a Master Mix

22 Why would you use a high fidelity polymerase?

23 High Fidelity Pfu DNA Polymerase Accuracy approximately 6-fold greater than Taq 3 5 exonuclease (proofreading) activity Robust activity

24 Is there any method, software or script that we can use that could help us predict potential nonspecific, but "sufficiently homologous" binding sites for specific primers in given sequences or sequenced genomes?

25 Primer Design Length: bases GC-content of 40 60% Avoid: three G or C residues in a row near the 3 -end self complementation secondary structure qpcr is different! (see MIQE guidelines for more details)

26 NCBI Primer Blast

27 NCBI Primer Blast

28 NCBI Primer Blast

29 NCBI Primer Blast

30 NCBI Primer Blast

31 NCBI Primer Blast

32 Primer3Plus

33 How can I calculate the best annealing temperature for my primers?

34 BioMath Calculator, T m Calculations for Oligos

35 Why would nested primers work, but not primers for qpcr?

36 Nested PCR

37 Why don t I have any bands in my gel?

38 Magnesium

39 How can we avoid inhibitors to PCR in whole blood samples?

40 Sources of PCR Inhibitors Reagents used for purification Avoid kits that use inhibitors Carryover from the sample Remove inhibitors if possible Lab equipment Inhibitor bile salts complex polysaccharides collagen heme humic acid melanin and eumelanin myoglobin polysaccarides proteinases calcium ions urea hemoglobin, lactoferrin immunoglobin G (IgG) indigo dye Source of Inhibitor feces feces, plant material tissues blood soil, plant material hair, skin muscle tissue plants milk milk, bone urine blood blood denim

41 Dealing with Inhibitors Choose reagents wisely Purification Polymerase Reduce, Remove, Resist, Dilute Spike sample into a positive control to test for inhibition Proper controls! Internal positive control IPC No template control NTC

42 What's the best method to calculate the DNA quantity of a PCR product from an electrophoresis gel? Is it feasible to infer it from an UV photo?

43 DNA Quantitation Ladder ng 50ng 25ng 12.5ng

44 Why do we sometimes see unexpected bands?

45 Unexpected bands Polymerase concentration: units of Taq DNA polymerase in a 50μl amplification reaction Template: Reactions with too little DNA template will have low yields, while reactions with too much DNA template can be plagued by nonspecific amplification. If possible, start with >10 4 copies of the target sequence to obtain a signal in cycles, but try to keep the final DNA concentration of the reaction 10ng/μl. Additives: BSA reduces the affects of many inhibitors like heme, tannic acid, humic acid etc Other additives often used include DMSO (1 10%) and formamide (1 10%) to reduce secondary structure issues

46 Unexpected Bands

47 Thank you!