MarcantonioLabProtocolManual——2

Quantitation of DNA

Detection of Nucleic Acids Using Absorption Spectroscopy 

The absorption of the sample can be measured at several different wavelengths to assess the purity and concentration of nucleic acids, as follows: 

1) Turn on the UV source on the spectrophotometer to allow it a few minutes to warm up before measuring absorbances to prevent drift.
2) Pipet 2 ul of the nucleic acid sample into an Eppendorf tube containing 498 ul of DD-H2O. Mix this and transfer it to a clean cuvette. Pipet 500 ul of DD-H2O into another cuvette that will be used as a reference sample. 
3) Place the reference cuvette into the instrument, close the lid, and set the wavelength to 260 by pressing the numbered keys 2,6, and 0 followed by the l key. 
4) Calibrate the instrument by pressing the Cal key. Wait until the readout indicates 0 absorbance and replace the reference cuvette with the sample cuvette. 
5) Close the lid and read the absorbance. 
6) Repeat steps 3, 4, and 5 for the wavelengths 280 and 325. 
7) When you are finished taking measurements, turn the UV source off and clean the cuvettes using the cuvette washer. 

Use the A260 to determine the amount of DNA present by the following formula:

Use the A260/A280 ratio and the A325 to estimate the purity of the nucleic acid sample as follows:

Absorbance at 325 suggests contamination by particulates/dirty cuvettes.

Purify/Concentrate-SQ

Purification and Concentration of Aqueous DNA Solutions for Plasmid DNA to be sequenced 

1) Add an equal volume of Phenol/chloroform to the sample to be purified and vortex vigorously for 10 seconds. 

If the volume of the DNA solution to be purified is below 100 ul, it is easier to perform the extraction step if the volume is first brought to 100 ul by adding TE buffer or H2O. 

2) Spin for 15 seconds at room temperature in a microcentrifuge. If the phases are not well separated, spin for 1 minute longer. 
3) Carefully remove the upper aqueous layer containing the DNA and transfer to a fresh microcentrifuge tube. 
4) Add 2/3 rd volume of 5M NH4OAc to the DNA solution, mix by vortexing briefly, and add 2 volumes of ice-cold ethanol. Mix by vortexing and place in crushed dry ice for at least 5 minutes to allow the DNA to precipitate. 
5) Spin for 5-10 minutes in a microcentrifuge. If the concentration of the DNA is believed to be low, or the size of the fragments is very small, more extensive centrifugation may be required. 
6) Carefully pour off the supernatant or aspirate it off, being very careful not to remove pellet. 
7) Add 1 ml 70% ethanol and invert several times to wash pellet. Spin as above. 
8) Dry the pellet in a Speed-Vac concentrator. 
9) Resuspend the pellet in an appropriate volume of H2O or TE buffer for further manipulations or storage. If this DNA is to be sequenced using the Sequenase Version 2.0 Kit, then the pellet should be resuspended in 20 ul H2O. 

Sequencing DS-DNA

Sequencing Double-Stranded DNA Using the Sequenase Version 2.0 Kit 

1) Prepare the DNA for sequencing as per the protocols, "Purification and Concentration of Aqueous DNA Solutions" and "Alkali Denaturation of Supercoiled Plasmid DNA". 
2) Warm a heating block up to 65 degrees. 
3) For each sample to be sequenced, put the following in a 1.5 ml Eppendorf tube:

• DNA (prepared as discussed) 7 ul

• Sequencing primer 1 ul

• Reaction Buffer 2 ul

4) Heat to 65 degrees for 2 minutes, then remove heating block from heat source and allow to slowly cool to <35 degrees. Heating the DNA separates the strands and allows the primer to anneal as the temperature slowly declines. 
5) While cooling, prepare the following mixtures. Prepare slightly more than you actually need, in order to avoid problems during the actual procedure. 

Dilute Labelling Mix: need 2 ul per sample

• 2 ul Labelling Mix (USB)

• 8 ul DD-H20

Dilute Sequenase: need 2 ul per sample

• 7 ul Enzyme Dilution Buffer (USB)

• 1 ul Sequenase 2.0 (USB)

Master Mix: need 5.5 ul per sample

• 0.1M DTT (USB) 1.0 ul

• (35S) dATP 0.5 ul

• Dilute Labelling Mix 2.0 ul

• Dilute Sequenase 2.0 ul

The Sequenase enzyme should not be removed from the freezer, if at all possible. It should be diluted and added to the Master Mix just before it is needed. The Master Mix can usually be prepared directly in the tube containing the 35S dATP. This minimizes the amount of contamination. 

6) Also at this time, prepare the microtiter dish that will be used during the termination reactions. Transparent tape should be applied over any wells that will not be needed, in order to provide a better seal against water leaking into the sample wells. The horizontal rows should be labelled A-T-G and C, while the vertical columns should be labelled with a code for your samples. When this is complete, place 2.5 ul of the appropriate ddNTP Termination Mix into each respective well. Use the appropriate set of Termination Mix tubes for either dGTP reactions (red-capped tubes) or for dITP reactions (green-capped tubes).

Be sure the drop of Termination Mix makes it to the bottom of the well by tapping the plate upon the bench. Keep the plate loosely covered with a plate sealer until needed. 

7) Once the annealed template-primer has reached 35 degrees or lower, add 5.5 ul of the Master Mix by pipetting it onto the side of the sample tube and tapping it down to mix thoroughly. Before adding the Master Mix, it is a good idea to briefly spin down the samples to recover any solution clinging to the top or sides of the tube.

Try not to contaminate the pipet tip with any sample, as it can be used to add Master Mix to all sample tubes, minimizing the amount of solid radioactive waste generated. 

8) Incubate at room temperature for 1 minute to allow for primer extension and labelling. 
9) Pipet the labelling reaction up and down to mix, and add 3.5 ul to each of the four wells on the microtiter plate containing the four termination mixes. One pipet tip can be used to add the reaction mix to the four wells by pipeting the sample onto the side of the well. The plate is then tapped on the bench causing the labelling reaction to mix with the termination mix. This procedure, once again, minimizes the amount of solid radioactive waste generated. 
10) Once all the reactions have been added to the termination mixes in the plate, cover the plate tightly with a plate sealer and place in a water bath at 37 degrees for 5 minutes.

The water in the bath should be high enough to bubble around the wells when you look under the plate. During this step the chains are extended a little more and then terminated by the addition of a dideoxynucleotide.

11) Wipe off the water from the outside of the plate with a Kimwipe and carefully remove the plate sealer, making sure that no drops of water fall into the wells. 
12) Add 4 ul of Stop Solution to each well by depositing the drop on the side of the well and tapping to mix. 
13) Samples may be stored, covered, at -20 degrees for up to 1 week before running on a gel.