So say that for an experiment which requires a [DNA] / [Protein] = 1.2 / 1 ratio, I have calculated that I need 96.2296 uM of duplex DNA.
This double stranded DNA is formed by annealing a template DNA strand which is 13 bases long and a primer DNA strand which is 13 bases long. This yields double stranded DNA with 9 bases that pair together and 4 bases overhanging on the 5' end of each strand.
If I ordered PCR grade DNA (I want to save money), I hear it's not that pure. I predict I would need to purify the DNA, anneal the template and primer strands of DNA with heat, separate the double stranded DNA on a gel, and then extract the doubles stranded DNA on a gel.
How much concentration template and primer DNA would I need. I will lose concentration purifying the DNA, forming the duplex, and extracting the DNA on a gel.
Anyone have any protocols for this?
only 1 micro liter, can be use less then this but not more than 1 micro liter.
Hello Adron,
The concentration of DNA to start must be 1microgram/mol. If it is diluted, and you do not have desired concentration. You can evaporated the elution buffer by putting it at thermostat at may be 80'.
It is always better to mix protein and DNA separately at the end and ratios should be protein =1M DNA =1.2M - 1.5M.
It means if you are adding 100ng DNA then protein concentration should be 0.85ng.
I hope it helps you.
You will need 5-10 mgs for crystallization. We used to purify by HPLC or ion exchange chromatography for smaller pieces of DNA like yours. Crystallization would be performed at ~10 mg/mL.
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