Sequencing a 10kb length of dna in a 12kb plasmid - (Apr/21/2007 )

Has anyone done a sequencing reaction on to sequence a 10kb dna in a 12kb plasmid before using a BigDye terminator?
Any comments on the extension time???

Any opinion or suggestion would be highly appreciated!

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I have done so for several constructs in the past.
No big deal if your sequencing primers are good. The length per reaction that's readable is a bit shorter than usual, so make sure you have a primer every 400 bp (if possible, if not, go antisense).
I used standard extension time, though I don't understand your question about this. It's impossible to do this entire sequence in one reaction, so increasing duration of extension time won't matter, it's just more reactions (I know this is expensive, but that's just the way it is).

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the cycling condition that my lab uses

melting temperature : 96 Celsius (30 sec)
annealing temperature: x celsius (15 sec)
extention temperature : 60 celsius (4min)

25 cycles

X ul of DNA (200 – 400 ng) DNA must be suspended in distileed water and volume must not exceed 5ul
2.0ul of 5X buffer
1.0ul of Big Dye (1ul for plasmid DNA, 0.5ul for PCR product)
0.5ul of Primer (10mM)
Y ul of sterile water (volume must be below 5ul)
10 ul Total Volume


3. Precipitation Protocol

90ul SD water
10ul Sodium Acetate (3M)
200ul Ethanol (100%)
1ul Pellet Paint


4. Leave at -20C for 30mins
5. Spin 13000 RPM, for 12min at 4C.
6. Remove supernatant. Pellet is blue in colour
7. Wash with 300ul of 70% Ethanol. Spin 12min at 13000 RPM, at 4C.
8. Remove supernatant. Pellet is blue in colour.
9. Spin 13000 RPM, for 3min at 4C.
10. Remove supernatant. Pellet is blue in colour.
11. Dry for 5 min.


Using this protocol I can get sequence reads with a minimum lenght of 700bp, and frequently 900bp. However there must be alot of black magic involved. When I first tried my hand at this, me sequence reads were about ~400bp. And my labmate uses this exact same protocol and he regularly gets a sequence read of 1100bp often ~1200bp. The subsequent clean up is as important (if not more so) then the actual sequencing reaction itself.

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Because there's an institute that offered sequencing of plasmid or something. i emailed the prof in the institute and here's the reply. i Don't know if he got the question entirely correct or not.

Post
"I need to sequence a plasmid(12kb) that has a transposon(2kb)
insertion. The actual gene of interest was disrupted by the
transposon insertion and is located somewhere in the 10kb of the
plasmid (12kb-2kb). If i provide to you the plasmid together with
the transposon Forward primer and reverse primer, can the
sequencing of the 10kb dna be done? This is not a sequencing of the
whole plasmid. that's why i would like to ask you for your opinion
before proceeding to the ordering."

Reply
"It should be quite easy to get sequence from transposon-specific
primers within a 12 kb plasmid. Spend some time designing appropriate
controls, however: for example, consider control primers that
sequence the plasmid from another position or two (e.g. T7, T3, SP6,
M13F/R), a control template with the transposon in a known position,
etc etc."

So anyway, my primary plan is to use this method designed by epibio(link below). It's an unknown plasmid with only one thing for sure, it has a Tn5 insertion confirmed by PCR reaction.

http://www.epibio.com/item.asp?ID=403&...mp;SubCatID=131

Thanks!
Here's the link . Let me know if this would be a good idea or not

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