1. Template : PCR or plasmid :

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Anthony Weaver
5 years ago
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500-1000bp : 5-20 ng 1000-2000bp : 10-40 ng > 2000bp : 20-50 ng Difficult to

1 1. Template : PCR or plasmid : + Plasmid : 200 ng in sequencing reaction needed + PCR product : depend on the size : bp : 1-3 ng bp : 3-10 ng bp : 5-20 ng bp : ng > 2000bp : ng Difficult to measure with spectrophotometer (nanodrop) Easy estimation : on gel : compare with ladder

2 + PCR product : cut out multiple bands : - Separate the fragments on an agarose (with 1 mmol/l guanosine) gel. - Place the gel on an UV source without turning on the UV. - Remove most of the buffer from the top of the gel with some tissue. - Turn on the UV-light and cut out the band as soon as possible (do NOT leave the light on longer than necessary!). - Make a small hole in the bottom of a 0,5 ml tube with a needle, and push a little filtration paper in the bottom of the tube. - Transfer the gel slice in this tube and freeze for 10 minutes at -20 C.

3 - Place this tube in a 1,5 ml one and wait until the gel slice is defrosted. - Centrifuge 1 minute at rpm. - The liquid collected in the 1,5 ml tube is the electrophoresis buffer with about 90 % of the PCR band. - Determine the volume and add 2,6 volumes (95 % ethanol + 0,12 M NaAc), mix and put at roomtemperature for 15 minutes. - Centrifuge 15 minutes at rpm (4 C).

4 - Remove supernatans (pellet is invisible) and add 125 µl 70% ethanol, mix gently and centrifuge 5 minutes at rpm (4 C). - Remove supernatans (pellet is invisible) and dry. - Add destilled water (for approximately the same concentration as in the PCR mixture, add the same volume as you used for loading the gel).

7 2. Sequencing protocol for Bigdye 3.1 on ABI 3130XL: + Cleaning PCR product : kits or enzymatic 1,25 µl cleaned PCR product needed for each sequencing reaction. If you have to sequence several reactions of one PCR product, adjust the volumes. For each reaction : pipet into a reaction tube : - PCR product : 5 µl - ExonucleaseI Fast AP thermosensitive Alkaline Phosphatase : 1 µl Mix, spin down and incubate 15 minutes at 37 C, followed by 15 minutes at 85 C to inactivate the enzymes. + Cycle sequencing Bring into a reaction tube the following products : - Terminator Ready Reaction Mix : 4 µl - cleaned PCR product : 1,25 µl (30-90 ng PCR product or ng double-stranded DNA) - primer (5µM) : 0,5 µl - sterile water : add until total volume is 10 µl

8 cycle sequencing : 1 min 96 C, 29 cycles : 10 seconds 96 C - 10 seconds 50 C 75 sec 60 C + Precipitation - add 26 µl (95 % ethanol + 0,12 M NaAc), mix and put at roomtemperature for 15 minutes - centrifuge 15 minutes at rpm (4 C) - remove supernatans (pellet is invisible) - add 125 µl 70% ethanol to each tube, mix gently - centrifuge 5 minutes at rpm (4 C) - remove supernatans (pellet is invisible) - dry 15 minutes under vacuum (These samples can be stored at -20 C) + Loading of the samples - add 10 µl Hi-Di formamide to each tube - mix well - transfer formamide from each tube to an 96 well plate - heat the samples for 2 minutes at 95 C - put the plate in the sequencer and run

12 3. Troubleshooting : + Double peaks : - contamination -> redo PCR with other primers, cloning - primer fits on more places -> use other primer - check if you had double peaks in PCR -> cut out bands

other - sometimes possible to fix by sequencing with

13 + Double peaks : possible heterozygote for that gene : - indication : 2 or more times the same peak next to each other - sometimes possible to fix by sequencing with internal primers, or from other Direction. If not : cloning

14 Heterozygote on a few positions in the gene :

15 + Strong secundary structure in the fragment : - seen around C's and G's : decrease of signal hight - sometimes additives in sequencing reaction helps : trehalose, betaine, DMSO

16 + bad resolution of the sequence : - problem with capillary -> replace by new - problem often occurs after fragmentanalysis -> extra refill of capillaries with polymer -> sometimes fixes itself after 1 or 2 sequencing runs

17 4. Example of fragmentanalysis on the ABI 3130 XL sequencer :

19 5. Assembling sequences to one gene : 6. Aligning the sequences :

20 Lots of alignment programs : ClustalW, Muscle, Mafft,...

21 7. Phylogenetics : + Based on one of the three methods : - Distance (Neighbor Joining, UPGMA) : pairwise distance -> distance matrix -> Clustering algorithm -> tree - Maximum Parsimony : most parsimonious tree is the one with the fewest evolutionary changes - Maximum Likelihood : calculates the likelihood for each tree using an explicit model of evolution + Determining the model of evolution : jmodeltest or MrModeltest + Lots of phylogenetic programs available : - Paup (uses NJ, MP, ML) - MrBayes : baysian interference (ML method) - Phylip - Molphy - Mega -...

22 - Branch length : number of changes that occured in that brach - Distance scale : % differences between 2 sequences Bootstrap values : % of calculated trees that species was found in that branch.

23 8. Next generation sequencing : - No experience (yet) - Expensive huge amount of data for that price (price per base is low) - Parallelized sequencing process - Very usefull for identifying outbreaks (E. Coli (EHEC)) and develop diagnostic kits. - Potential for phylogenetics - Potential for museum samples - Potential for heterozygotes -...

24 Heterozygote with next generation sequencing :

25 Jun Zhang, Rod Chidini, Ahmed Badr, Genfa Zhang, The impact of next-generation sequencing on genomics. Journal of Genetics and Genomics 38 (95-109).

26 Bybee S. et al Directed next generation sequencing for phylogenetics : an example using Decapoda. Zoologischer Anzeiger.

27 Ion Torrent Personal Genome Machine (PGM) :

28 Thank You Andy Vierstraete University of Gent Belgium

1. Collecting samples :

1. Collecting samples : + Preservation of samples is very important to conserve DNA + Preservation solutions and methods: * aceton (50-100%) (flammable) * ethanol (90-100%) (flammable) * 2-propanol (flammable)