Sequencing - (May/14/2007 )

Hi

I am having problems with sequencing.

I nanodroped my plasminds and the concentrations were in the range of 300-400ng/ul.

Then I prepared the sequencing reaction with Bigdye 0.5 ul (diluted stock), 5xBuffer 2ul, S-tag primer (I use pET vector) 0.5ul, DNA 1.5-2ul, water 4-4.5ul (to make it 10ul reaction).

I have repeated the sequencing twice with replicates but I did not get anything (all blanks) .

Please give me some suggestion to improve the sequencing as I am in a critical situation to proceed further.

Cheers

Ganes

Three things I can think of:
1. I do not 100% trust nanodrop quantification.
You probably want to run your gel with ladders to make sure you got plasmid in that range.
2. Check your thermocycling program for your primer....heat denaturating starts also would help.
3. cleanup after sequencing to remove unbound primers, BD, salts, etc.

the most important thing I found with sequencing, is not to use too much big dye (which seem okay in your case) and the subsequent clean up when you percipitate your sequencing reaction prior to sending it off.

The percipitation step is very important. Get it wrong and either you lose your pellet or get a fantastically horrible signal.

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 4Celsius.
8. Remove supernatant. Pellet is blue in colour.
9. Spin 13000 RPM, for 3min at 4 C.
10. Remove any additional supernatant. Pellet is blue in colour.
11. Dry for 5 min.

Thanks Perneseblue and Monard for your valuable comments.

I will try your suggestions.

Cheers

and one more things before I forget. Contamination with PEG is bad news for sequencing. If you conduct PEG percipitation on your DNA, you must throughly remove the PEG (by resuspending the pellet, followed by ethanol percipitation. Repeat. PEG is soluble in ethanol, but has difficulties disolving in ethanol if trapped in a pellet)

Make sure your DNA sample is free of phenol, RNA and secondary bands. Gel purify your sample (if PCR product) if there are secondary bands present.

I used another approach, though similar with perneseblue's

1. Add 2 ul 125 mM EDTA and 2ul 3M NaOAc, pH 5.2 into each tube.
2. Add the 10 ul dideoxy sequencing reaction product into each of the tube.
3. Add 50 ul absolute EtOH. Mix and precipitate at RT, 20 min, away from light.
4. Spin at max speed, 15 min at RT. (RT seemed to be just good to precipitate the dideoxy seq product while leaving unincorporated dye in suspension)

5. Discard s/n by pipetting (I dislike inverting the tube as liquid will stick to the wall of the tube). Overlay 250 ul 70 % EtOH onto the "invisible" pellet (I prefer overlay without vortexing). Spin at max speed at RT, 10 min. Discard s/n by pipetting.

6. Dry in a Speed Vac, or heat in heat block to evaporate residual liquid.
7. Add HiDi only when you're about to run sequencing. Store at -20 C for not more than a month (some mentioned 3 months).

Bests