DNA Sequencing

What to bring?

The following types of DNA are suitable for sequencing. Please provide the specified amount of DNA:

• Double stranded DNA - 500ng

• Single stranded DNA - 100ng

• PCR fragments - 10ng for every 100 nucleotides

Please notice:

• All samples in water

• 260/280 = (1.8-2.0)

• 260/230 = (1.6-3.3)

• A Thermo Scientific NanoDrop 1000 spectrophotometer is available at the Unit for testing samples of DNA for sequencing. (Please notice that due to extensive workload the use of this spectrometer is restricted only to DNA samples that are submitted for sequencing).

Primers

• Standard primers (T3, T7, etc). will be provided by the Unit

• Internal primers should be 18-25 bases with 50% GC (Tm=>50ºC)

• Please provide 10 pmol of primer

• Total volume of primers and sample should be 16ul

• Sequencing PCRs will be performed at 50ºC

What not to bring!!!!

• Samples with salt (260/230 is low)

• Samples with ratio 260/280 out of range

• Samples with RNA

• Samples with slow growing host vectors -jm101

• Samples with ethanol or phenol still in them

When to bring samples?

The DNA Sequencing Facility is open 5 days/week.

Samples should be handed in from 10:00-11:00AM.