dna sequencing problem - (Sep/18/2008 )
Hi, i just started at my new lab and is having problem with sequencing.
I transformed my gene of interest into E.coli, and then extracted the plasmid. From there i use the plasmid as template for cycle sequencing. However, my failure rate is extremely high. when i sent in 16 samples only 1 came back fine. Subsequently i sent int 4 samples and only 1 worked as well.
I tried doing PCR using the vector primers (T3/T7) and is able to see single clear band of approximate right size.
The reaction mix i used is 1ul of 1.6pM T3primer, 280ng plasmid, 4ul buffer, 2ul bigdye, then topped up with nuclease free water till 20ul. (I got this from my colleages in my old lab who had no problem with sequencing of their plasmid)
I have previously done lots of sequencing using purified PCR products as template at my old lab with no problem. Thus i feel that it is unlikely that i may have problem with preparation of samples and cleaning. Can anyone give me some suggestion. Could it be a problem of the company that is sequencing the sample for us?
Possibly. One of my colleagues had the same experience.
looks to me you are using too much big dye. how about trimming it down to 1ul.
Common problems with sequencing reaction I have notice so far are
-using too much DNA template
-using too much big dye
-PEG.
How are you purifying your plasmid? I had some problems sequencing plasmids from minipreps with the kit from fermentas - solved the problem by leaving the elutionbuffer in the column for 5 mins before centrifuging for the last time and by increasing the time to dry the column.
When you say that you fail, then what do you mean by it? Usually the cause is too much DNA or problems with purity. In the trace files of the failed samples, do you see very tall curves in the start of your file and drastic drop off afterwards? This implies at too high DNA concentration or some residues left after EtOH washes (capillary got clogged). Solution would be to take less DNA and wash longer with EtOH. Often it would help to run the same failed sample again. Different companies have their sequencing machines adjusted for different settings. You could call them and ask for advice.
I transformed my gene of interest into E.coli, and then extracted the plasmid. From there i use the plasmid as template for cycle sequencing. However, my failure rate is extremely high. when i sent in 16 samples only 1 came back fine. Subsequently i sent int 4 samples and only 1 worked as well.
I tried doing PCR using the vector primers (T3/T7) and is able to see single clear band of approximate right size.
The reaction mix i used is 1ul of 1.6pM T3primer, 280ng plasmid, 4ul buffer, 2ul bigdye, then topped up with nuclease free water till 20ul. (I got this from my colleages in my old lab who had no problem with sequencing of their plasmid)
I have previously done lots of sequencing using purified PCR products as template at my old lab with no problem. Thus i feel that it is unlikely that i may have problem with preparation of samples and cleaning. Can anyone give me some suggestion. Could it be a problem of the company that is sequencing the sample for us?
I saw this paper a few weeks ago in Biotechniques. See what you think. http://biotechniques.com/article.asp?id=112902
You can also ask the company doing the sequencing runs for you what advice they have.
Common problems with sequencing reaction I have notice so far are
-using too much DNA template
-using too much big dye
-PEG.
I had the same problem when i used less bigdye =(
I'm purifying my plasmid using miniprep kit. I always leave the elution buffer for approximately 5 minutes before centrifuging, and i dry the column for 2 mins before elution
I don't think there is a problem with purity of the plasmid because i've used nanodrop to check its concentration and 260/280 ratio. I do not see tall curves dropping drastically.
When i mean fail i mean that the analysed trace shows low noisy peaks overlapping all over, with random base calls. And the Raw trace shows almost no peaks, with the 4 colour dyes not having the same baseline.
I have talked to the company but they said they are not having problem with the other samples. They are suggesting that i have problem with my cleanup, that i may have over-dry my sample at the last step of cleanup.
But the most frustrating thing is that i have prepared 2 samples the same way i prepared for the company's, and sent it to another place for sequencing. Both the samples gave very clean nice peaks.
