12 replies to this topic

[lizrene]

Hi there,

Having a problem here in big dye. I am doing my cycle sequencing using the PCR purified product (after gel-slicing). However, the result which i get from the sequencing are short reads/noisy data. What causes the short read/noisy data. I insert: 1uL of my primer, 1ul of sequencing buffer, 2.0ul of template (300ng/ul), and 1ul of big dye. what should i do???

Lizrene

[phage434]

Short reads are usually the result of too much template DNA.  Sequencing reactions are one of the few places where both too much and too little DNA can cause problems.

[lizrene]

what about noisy data? my sequence has lots of 'N'

[phage434]

If the reads are good for a while, then drop in amplitude dramatically, then reporting N's, I'd suggest that the problem is too much DNA.  You should be looking at the trace file, not at the called sequence.  If you have the trace file (.ab1 file e.g.) then I could tell more.

[Maddie]

Can you show a picture? A negative control (or failed seq) will give you unreadable sequences with lots of N. You could also have too much background because you injected too much sequence.
It's hard to tell without seing the electropherogram.

[lizrene]

phage434, on 20 April 2011 - 07:41 PM, said:

If the reads are good for a while, then drop in amplitude dramatically, then reporting N's, I'd suggest that the problem is too much DNA.  You should be looking at the trace file, not at the called sequence.  If you have the trace file (.ab1 file e.g.) then I could tell more.

HI,

As attached ...  SeqResults38294.zip   600.54K   76 downloads

[lizrene]

Maddie, on 21 April 2011 - 07:48 AM, said:

Can you show a picture? A negative control (or failed seq) will give you unreadable sequences with lots of N. You could also have too much background because you injected too much sequence.
It's hard to tell without seing the electropherogram.

Hi maddie,

As attached. Thanks

Lizrene

Attached Files

•  SeqResults38294.zip   600.54K   76 downloads

[phage434]

I'd say that you either have no template DNA (have you verified on a gel?  Tried restriction digests?) or that your primers are not binding.  There is no interpretable sequence data in those files.  How was your DNA prepared?  Primers?  How do you know that the primers will bind to your template?

[Maddie]

I agree with phage. A PCR with too little or no DNA would look like that. Another possibility is that you used too much DNA. 600ng from PCR product is far too much. I attach the manual of the kit, see page 2-6. For PCR product, it doesn't go higher than 30 ng.
How did you quantify your DNA?

Attached Files

•  seq kit manual.pdf   564.69K   126 downloads

[lizrene]

phage434, on 22 April 2011 - 04:17 AM, said:

I'd say that you either have no template DNA (have you verified on a gel?  Tried restriction digests?) or that your primers are not binding.  There is no interpretable sequence data in those files.  How was your DNA prepared?  Primers?  How do you know that the primers will bind to your template?

Hi phage 454,

To verify on a gel, is it after the Big Dye? If after the big dye, i did not do it. I did not use any restriction digests. My template is from a PCR Purified Product. I did use the same primers like i did for my pcr amplification. it worked cause i get my targeted size band.what do you suggests? I did the big dye manually. And the protocol as follow:

After the Big Dye (5ul)- 40 cycles,I add 1ul of 125mM EDTA, 1ul 3M NaOAc (pH 4.6), 25ul 100% Ethanol, where all these are reached to the bottom of the tube.I tab to mix, incubate at room temperature for 15 minutes.Spin at max speed 13200 rpm for 20 min. I discard the supernatant using pipette. I add 35ul of 70% ethanol (prepared fresh). again spin max speed, 13200 rpm, 10min at 4C. Discard the supernatant. Add 35 ul of 70% ethanol, spin 1 min, discard the ethanol. Dry at 65C for 20 min in incubator. Finally wrap with the aluminium foil. This is what i did.

[lizrene]

Maddie, on 22 April 2011 - 10:20 AM, said:

I agree with phage. A PCR with too little or no DNA would look like that. Another possibility is that you used too much DNA. 600ng from PCR product is far too much. I attach the manual of the kit, see page 2-6. For PCR product, it doesn't go higher than 30 ng.
How did you quantify your DNA?

hi maddie,

thanks for the manual kit maddie. But i did the cycle sequencing manually. Is it ok if i use 30ng? i quantify using spectrophotometer. if my concentration is higher, than i dilute them using m1v1=m2v2 formula.

[perneseblue]

On the goldilocks subject of too little or too much DNA,

Maybe this might be useful.
I use 1ul of Bigdye in a 10ul reaction for sequencing plasmid DNA. 0.5ul bigdye for PCR product
The amount of DNA used is 25ng * kb (DNA size)

[EHMehtyl]

I am new in the field and saw this posts, I was planning to sequence my PCR product with normal sequencing and was told it has to be pyrosequencing. I can't understand why? So you are amplifying your products with bigdye? I wouldn't like to spend so much money in biotinylated primers! What would be the advantage of pyrosequencing?  Thanks!!