Miniprep for sequencing - (Dec/01/2006 )
I am wondering if anyone used miniprep products for sequencing... in my hand, miniprep never produce concentration that is high enough, so i always use either midi or maxiprep. I followed exactly the Qiagen's protocol, but still the miniprep concentration is low (it is said about 0.5-1ug/ul though).
Another concern is, the miniprep quality is not as good as maxiprep's since it does not have isopropanol precipitation?
Thanks!
Another concern is, the miniprep quality is not as good as maxiprep's since it does not have isopropanol precipitation?
Thanks!
I have done it a couple of times, on miniprep from colums and minipreps with alkaline lysis. I never had problems with it, you can include a phenol-chloroform extraction (if that is not in your common protocol).
Off course I don't know what you want to sequence, but for sequencing your insert for instance, you don't need much DNA.
The place were we send our samples for sequencing wants DNA extracted using a kit and I used to do it. But then, I started to send samples extracted using standard protocol (without saying anything 
) and it always work fine!!
Bay the way, it supposed DNA should be at 1-2 ug/ul, but some times I send samples at 0.2-0.5 ug/ul (without saying anything ) and again, it always work perfectly fine!
2 ul culture Over night,
100 ul sol I (with RNasa), 200 ul sol II, 150 ul sol 3. centrif
phenol-chloroform extraction.
Pp 0.7 V Isopropanol. Wash EtOH 70 %.
Final vol: 30-50 ul.
500 ng - 1 ug of plasmid DNA is heaps for a sequencing reaction. Of course it also depends on the size of your vector. The larger the vector, the more DNA you need to add. I have sequenced from a fosmid vector containing a 40 kb insert using the Qiagen miniprep to purify so it shouldn't be a problem. I would give it a go as Azteca Princess has said, you might be surprised.
Thanks all. I did but actually the result is bad... I assume the concentration is too low.
500 ng - 1 ug of plasmid DNA is heaps for a sequencing reaction. Of course it also depends on the size of your vector. The larger the vector, the more DNA you need to add. I have sequenced from a fosmid vector containing a 40 kb insert using the Qiagen miniprep to purify so it shouldn't be a problem. I would give it a go as Azteca Princess has said, you might be surprised.
Thanks all. I did but actually the result is bad... I assume the concentration is too low.
I don't think the problem is the concentration.
Good luck
Is the RNA in the final solution a probem? Do you resuspend in TE+RNase or just water?
) and it always work fine!! Bay the way, it supposed DNA should be at 1-2 ug/ul, but some times I send samples at 0.2-0.5 ug/ul (without saying anything
) and again, it always work perfectly fine!2 ul culture Over night,
100 ul sol I (with RNasa), 200 ul sol II, 150 ul sol 3. centrif
phenol-chloroform extraction.
Pp 0.7 V Isopropanol. Wash EtOH 70 %.
Final vol: 30-50 ul.
RNA in the final soution could be a problem for sequencing. We use columns for pure grade DNA. I would elute in water for sequencing.
"Thanks all. I did but actually the result is bad... I assume the concentration is too low."
Why is that? What do your chromatograms look like? Any chance of a pic, or a description?
Never faced a problem eluting in Tris. We use Qiagen miniprep kit and get good results. TE might give problems with the EDTA present, but as we only use 1 µl in a 10 µl sequencing reaction it most likely wouldn't matter either.