FAQ

FAQ's for Sequencing


Q: What kinds of templates can you sequence?

A: We can sequence plasmids, PCR products, phage and some BAC templates. Please keep in mind that PCR products need to be purified away from the amplification primers and any spurious amplification products. We can provide purification services, if desired. We ask that templates be provided in an EDTA-free buffer such as water or 10mM tris.

Q: What amount of template and primer do you need?

A: We will need ~35 ng of template DNA per 1Kb of total template size. Therefore, if we are performing a reaction on a 3Kb vector with a 2.5Kb insert we would need ~180 ng for that reaction (5.5Kb x 35ng). Likewise, if we were performing a reaction on a 350bp PCR fragment, we would need ~12 ng of template DNA. For each reaction we use 1ul of a 10uM primer dilution. Primers can be supplied at a higher concentration, as long as it is clearly marked.

Q: How many bases do you get per reaction?

A: We normally get between 450 and 650 bases of quality sequence per reaction. Please keep in mind that this can vary due reasons including template quality, sequence composition and many others.

Q: How quickly will I get my results?

A: Our normal turn around time is one to two business days.

Q: Do I need to provide the sequencing primers?

A: We have most common vector primers here at the lab, so unless you require gene specific primers, you do not need to provide them. Please keep in mind that Lofstrand does offer Oligonucleotide synthesis services, so even if you do need custom primers, we can do that for you as well.

Q: Can you directly sequence genomic DNA?

A: We do not directly sequence genomic DNA. However, we can amplify a target region as a PCR product using a high-fidelity polymerase and use that product as a sequencing template.

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FAQ's for Plasmids


Q: How do you isolate your plasmids?

A: Routinely, we isolate the plasmids using CsCl gradients. This procedure yields the best quality plasmids that are ideal for labeling, transfection, RNA transcripts, and other molecular biology steps. It also yields more DNA than using other methods. We also isolate plasmids using affinity columns.

Q: What do we need to send ?

A: If it is a bacterial culture, we prefer to receive it on a fresh plate culture. But we accept cultures in the form of fresh overnight, stab, or glycerol freeze. We also accept DNA. There will be additional charges for transformation and miniprep check when the starting material is DNA.

Q: Can we order by the mg.

A: It is not possible to predict the yield of DNA from bacterial cultures. We therefore charge by the liter of bacterial culture, not by mg.

Q: Do you isolate other vector DNA?

A: Yes, we also isolate BACs, PACs, fosmids, Cosmids and dsM13.

Q: What is the turnaround time?

A: 5-7 working days from culture, 10 working days from DNA.

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FAQ's for Oligonucleotide Synthesis

 

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FAQ's for Labeled Probes

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FAQ's for Nucleic Acid Isolations


Q: How do you isolate genomic DNA and from what type of starting sources?

A: Routinely, we isolate genomic DNA from blood, tissue(plant/animal/microbial) and cell culture and purify the DNA by proteinase K treatment and ammonium acetate/SDS and/or phenol/chloroform extraction. This procedure yields the high quality DNA that is ideal for labeling, restriction digests, Southern blots, genomic libraries, and other molecular biology steps.

Q: How do you isolate RNA and from what type of starting sources?

A: Routinely, we isolate total RNA from blood, tissue(plant/animal/microbial) and cell culture and purify the total RNA by Trizol extraction. This procedure yields high quality RNA that is ideal for labeling, Northern blots, and other molecular biology steps. Poly A RNA is purified using oligo dT affinity columns.

Q: What do we need to send ?

A: If it is a bacterial culture, we prefer to receive it on a fresh plate culture. But we accept cultures in the form of fresh overnight, stab, or glycerol freeze.

Blood, tissue culture cells, animal tissue, plant tissue, and microbial cells can be sent frozen on dry ice or in RNA later or RNA Ice(Ambion).

Q: What is the turnaround time?

A: 5-7 working days