Sample preparation

Sample preparation for sequencing reaction and analysis:

Prepare samples in 0.2 mL thin-walled microtubes, strips or in a plate. Use your abbreviation and number from the Sample sheet to label the tubes; strips can be numbered whole, but always so that there is a clear orientation from sample 1 - 8. Just name the plates with the abbreviation of your name and the date, but make sure the wells are sufficiently sealed - watch out for leaking foils. Pipette and number by column!

The total volume for a one-way sequencing reaction is 8 μl:

-primer - only ONE at amount of 5 pmol (we recommend taking 1 μl from the stock 5 uM primer solution). Tm 50-55°C opt., Tm 45-72°C max. (always note Tm different from the optimum in the Sample sheet, as well as the use of degenerate primers - but their use is rather not recommended). We will adjust the profile of the sequencing reaction. Some universal primers can be added to the reaction, than the sample volume is 7 μl.

- DNA in the amount of 5 ng/100 bp for PCR products and 3 ng/100 bp of plasmid DNA (the full length of the plasmid is counted, usually corresponding to 100-200 ng in the reaction, max 300 ng for large plasmids). Sequencing of small fragments and large constructs (over 12 0000 bp) should be discussed with the laboratory in advance. The recommended amount is a guideline, high quality and pure DNA can be sequenced from even a small amount, on the other hand too much DNA can block the sequencing reaction. If the sequencing is going well, of course do not change the concentration!

For preparation of samples purified with ExoSap and samples isolated from the gel, see here.

- water (good quality) or TRIS, never TE buffer - EDTA blocks the reaction!!!

Price per one reaction and analysis: for Charles University - 87 CZK, for other academic institutions - 98 CZK, for private entities - 100 CZK.

Sample preparation for Hairpin protocol:

If the sequencing polymerase falls off on DNA hairpin, we recommend using additives and the Hairpin protocol. Prepare two tubes - separately 2 ul of primer at 5 pmol and separately 4 μl of DNA - total amount of 150 ng.

There is an additional charge of 20 CZK for this protocol.

Samples praparation for Analysis of purified sequencing products:

Deliver samples in 0.2 or 0.5 ml tubes, properly dried. Ethanol residues will interfere with sequence analysis. Dry in an open tube in approximately 5 min at 50-60°C. Samples should be supplied in an opaque bag.

Price per analysis: for Charles University - 33 CZK, for other academic institutions - 39 CZK, for private entities - 40 CZK.