Plasmids must be brought to us in water at 0.2µg/µl concentration. Concentration should be determined by O.D. 260. This is the most accurate. Quantitation via gel can be off by as much as 4 times. Incorrect concentration is the main cause of failed sequencing reactions. Accurate concentration greatly improves the quality of data. DO NOT bring your samples in TE. EDTA chelates magnesium, and Taq has an absolute requirement for magnesium, so the sequencing reactions will not work if the samples are in TE. However, you may use modified TE.
Primers should be in water at 1µM concentration. Please do these calculations and make the dilution yourself. If you have a primer synthesized in our facility that will be used for sequencing, you may check the "Please give oligo to DAF for sequencing" box on the oligo request .
Both double-stranded template and primer must be brought mixed together in a single 1.5 ml microfuge tube - 12 ul of 0.2 ug/ul plasmid and 12 ul of 1 uM primer. If you do not bring them as requested, there will be an additional charge of $5.00.
We recommend the following purification methods:
The following purification methods may be used, but are not recommended:
The above procedures work very well in some people's hands, but not everyone's. To use them you must be meticulous, being sure to remove all protein (O.D. 260:280 should be 1.8. If it is any higher then you have protein left and must re-extract or expect "noisy" sequence. If it is lower then you have RNA present and you should re-RNAase. RNA does not typically ruin the reaction, but it interferes with quantitation and inaccurate quantitation can ruin a sequence.) Care must be taken when using Phenol-Chloroform and Cesium Chloride extraction protocols as both residual Phenol and Cesium will interfere with the sequencing reaction if they are left behind in your prep.
The following purification methods should be avoided:
In our experience,the above give poor or inconsistent results. Gene-Clean uses Sodium Iodide, which can interfere with the sequencing reaction. Wizard mini-preps have shown, in our hands, to give inconsistent yields and purity.
Some tips for longer and more accurate sequence:
If you are bringing us the template because you have been unable to sequence it in the past, please let us know. We may be able to use special protocols that will help increase the chance of successfully sequencing your sample.
If you know your template has a poly-A tail, consider using the oligo primer dt-V instead of the vector primer for that end. Poly-A tails will cause the Taq to slip and the sequence after the tail will be unreadable. The oligo dt-V helps this problem some of the time. If you know what the base after the tail is, use either the oligo dt-A, oligo dt-G, or oligo dt-C primer.