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Protocols

Preparing Template DNA for Sequencing

It is of vital importance that template DNA is well purified and accurately quantified in order to achieve successful sequencing reactions and obtain high quality data.

DNA Quality

The quality of DNA in a reaction can affect the performance of the DNA Analyser. Contaminants present in cycle sequencing reactions negatively affect polymerase binding, amplification or extension. This results in data with no usable sequence, low signal, high background noise or short read lengths (please see our troubleshooting page for examples).

When preparing template DNA it is critical to avoid the following:

There are various methods available to purify template DNA prior to carrying out sequencing reactions. These can be found in chapter 3 of DNA Sequencing by Capillary Electrophoresis Chemistry Guide.

We recommend the Acroprep 96 Filter Plate from the PALL Corporation for the purification of PCR products.

 

DNA Quantification

We recommend a combination of running your samples on an agarose gel and using a spectrophotometer/nanodrop/Qubit to quantify them, rather than relying on just one method.

 

 

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Sequencing Reaction Protocol

If you want to carry out your own reactions and then submit them for run only, we recommend carrying out 10µl reactions using the following volumes of components (per sample):

5 x Sequencing Buffer
1.75µl

Big Dye Terminator
0.5µl
Primer (3.2┬ÁM)
1µl
Template
0.5µl - 4µl*
Distilled Water
to bring total up to 10µl

* In a 10µl reaction we suggest using the following quantities of template:

PCR products
1ng/100bp
Plasmids
150ng for plasmids up to 4Kb, or 200ng for larger plasmids

 

Recommended cycling conditions for sequencing reaction

96°C 10s
50°C 10s
60°C 60s*
Repeat for 25 cycles
Hold at 4°C

* For 1000bp fragments (you may choose to increase or decrease the extension time depending on the size of your product). We carry out a 2min 30sec extension step as standard for all samples.

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Sequencing Reaction Clean-Up Protocol

We recommend the use of Sephadex 50 columns to remove unincorporated ddNTPs:

Reference

Zoon RA (1987) Methods Enzymol. 152: 25-29.

This method is based on gel filtration. The Sephadex resin is comprised of small beads, which have small holes in them. Big molecules such as sequencing products cannot fit into the holes and so they go around the beads and move quickly through the column when pressure (centrifugal force) is applied. Small molecules (dNTPs) fit into the holes in the beads and they move very slowly through the column and get retained. When the column is spun at low rcf for a short time, only the pure sequencing product is eluted and the dNTPs stay in the resin.

To prepare resin

  1. Weigh 25g Sephadex G-50
  2. Add 500ml ddH2O
  3. Mix well and stand about 10 minutes
  4. Suck cloudy water off to ~ 1cm above resin
  5. Refill with ddH2O
  6. Go back to step 3 and repeat until upper ddH2O layer is clear
  7. Store in the fridge

To use resin

  1. Suck water down to 1cm or so
  2. Mix resin well (no stir bars)
  3. Use

Protocol

  1. Set up two Whatman Unifilter plates with deep well collection plates underneath: one for clean-up and one for balance
  2. In one plate, aliquot 500µl of sephadex resin in the number of wells required (using a cut-off tip) In the other plate, aliquot equivalent weight in water
  3. Spin at 750xg for 5 mins
  4. Flick the water off the collection plates, rebalance, and spin again at 750xg for 3 mins
  5. Flick the water off the collection plates then add 250µl sephadex resin on top of the 500µl resin in the wells
  6. Rebalance and spin at 750xg for 5 mins
  7. Flick the water off the collection plates
  8. Replace the deep-well collection plate with an empty PCR plate and spin at 750xg for 1min
  9. Check the PCR plate. Some water should be present but not too much (<5ul per well) This shows that the resin in the plates isn't too wet or dry
  10. Quickly spin down your 10µl sequencing reaction and then add 10µl of ddH20 to each sample (you get better elution with 20µl sample volume)
  11. Apply your sample to the sephadex column (ideally, watch the sample "drop" onto the column: don't let it slip down the side and/or ram the pipette tip into the column)
  12. Replace the collection plate with a clean PCR plate for collecting your sample
  13. Balance and spin at 750xg for 5 mins
  14. Check the plate to ensure that the samples are collecting ok and then spin at 750xg for further 3 mins
  15. Check that the volume is close to 20µl (if not, bring it up to 20µl, don't worry if it's > 20µl)
  16. Seal the plate, wrap it in tinfoil, and label it well

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© Department of Anatomy
University of Otago
PO Box 913
Dunedin 9054
New Zealand

Email gas@anatomy.otago.ac.nz