Introduction to Arlequin 3.1 – Barber Lab (DD & EC)
1) Getting your Sequence Data Ready for Arlequin
2) Create a blank Arlequin project and import your data
3) Setup and Run your Tests
4) Analyze your results
Introduction to Arlequin 3.1 – Barber Lab (DD & EC)
Cycle sequence is similar to PCR. It uses most of the same ingredients, follows the same basic procedure, and is done in a thermal cycler as well. One key difference is that only one primer is used in each cycle sequencing reaction so that the amplification of product is linear, not exponential. Another key difference is that dideoxynucleotides are used which interrupts the extension of the DNA strand when incorporated. Remember that when doing cycle sequencing you will have 2 master mixes, and that only one primer goes in each master mix. Because cycle sequencing is a linear amplification process, it is less susceptible to contamination, but maintain good sterile technique anyway. Cycle sequencing must only be done in the HIGH DNA area of the lab. Use thermal cycler specified for cycle sequencing.
HOT START PCR (For manual Hot-Start, Double-Stranded Amplifications)
PCR will make about 40 Billion copies from a given template. Because of this, it is imperative to maintain excellent sterile technique throughout as any contaminant will be amplified along with (or instead of) your template DNA. Work in the LOW DNA portion of the lab.
Introduction to PAUP* – Barber Lab (PB & EC)
1) PAUP* and Modeltest
2) PAUP*’s Diverse Tree Search Methods
3) Bootstrapping
4) Kishino-Hasegawa Test
PCR product “clean-up” protocol: SAP/EXO
The purpose of this procedure is used to chew up excess primers and remove excess dNTPs from your PCR product. This proceedure is necessary to ensure clean and readable DNA sequences. Work in HIGH DNA part of lab.
Agarose Electrophoresis
E. Crandall/P.Barber
Agarose electrophoresis is performed to visualize your PCR products. This step allows you to determine whether your PCR was successful, whether the resulting product is the correct size, whether other products were amplified as well, and whether the concentration of the resulting product is suitable for cycle sequencing. All gel work should be performed in the HIGH DNA area.
DNA Sequencing in 10 Easy Steps
1. Separate and individually label samples for reference.
2. Perform DNA Extraction, labeling extractions to match above. Save in the
refrigerator.
3. Individually amplify 1-2uL of each sample via PCR, labeling amplifications to match above.
4. Electrophores 3uL of each PCR productand DNA ladder on an agarose gel. Save remaing PCR product at room temp.
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