|DNA Template Prep and Sequencing Reaction Cleanup|
|Montage Sequencing Reaction Cleanup|
|MultiScreen 384-SEQ Filter Plate; Ready-to-use, automation-friendly, 384-well plate for dye terminator removal|
|Gene-scan method for the recognition of carriers and patients with p47phox-deficient autosomal recessive chronic granulomatous disease.|
Jan Dekker, Martin de Boer and Dirk Roos; Experimental Hematology 29 (11): 1319-1325
Experimental Hematology 29 (11): 1319-1325 2001
|How much template should I add when using Montage SEQ96 Sequencing Reaction Cleanup Kit?||Irrespective of reaction scale, our suggestion is 50-150 ng of plasmid template or 5-10 fmol of PCR product template. The amount of template and primer present is proportional to the amount of sequencing product synthesized in the reaction. Do not proportionately scale down the amount of template and primer as you miniaturize.|
|When using Montage SEQ96 Sequencing Reaction Cleanup Kit, why do you suggest BigDye v. 3.0?||Our Phred and cross-match analyses consistently suggest that the use of BigDye v. 3.0 results in significantly longer, more accurate reads than any other dye terminator sequencing chemistry. Also, for those facilities where reagent cost reduction is an important consideration, BigDye v. 3.0 is more amenable to miniaturization than other dye terminator chemistries.|
|When using Montage SEQ96 Sequencing Reaction Cleanup Kit, why do I see low signal in one or more channel?||Dye blobs (i.e., unincorporated dye terminator peaks) are the most common cause of this artifact. The sequencing analysis algorithm attempts to normalize the signal intensity in all four channels, and if there is a large unincorporated dye terminator peak corresponding to a particular peak, all subsequent peaks of this type will be minimized in the final electropherogram. Examination of the raw traces will indicate whether this is the cause. Ordinarily, the data can be reanalyzed at a start point beyond the artifact peak to alleviate any interference with analysis.
See the FAQ "When using Montage SEQ96 Sequencing Reaction Cleanup Kit, why do I see dye blobs?" to permanently eliminate this problem.
|When using Montage SEQ96 Sequencing Reaction Cleanup Kit, why do I see slow filtration?||See FAQ "When using Montage SEQ96 Sequencing Reaction Cleanup Kit, why do I see dye blobs?"
Another potential cause of slow filtration is the presence of detergents in PCR buffers, if you are using PCR product as template. Triton X-100 is sometimes an additive to some manufacturers’ PCR buffers supplied with Taq or other polymerase
|When using Montage SEQ96 Sequencing Reaction Cleanup Kit, what are the optimal injection parameters?||ABI 3700-
injection solution injection- 2 kV for 15 sec- injection solution is the preferred injection vehicle.
Water injection- 1 kV for 10 seconds- This optimizes the peak spacing and resolution while maintaining signal strength. More aggressive injection can cause overloading and degrade sequencing quality.
injection solution injection- 2 kV for 30 sec- injection solution is the preferred injection vehicle.
Water injection- 2 kV for 10 seconds- This optimizes the peak spacing and resolution while maintaining signal strength. More aggressive injection can cause template clogging and degrade sequencing quality.
ABI 3100 and 3100Avant
injection solution injection- 1.5kV for 20 seconds- injection solution is the preferred injection vehicle
|When using Montage SEQ96 Sequencing Reaction Cleanup Kit, how should I set up my sequencing reactions?||Our definition of reaction scale:
A 1X reaction always uses 8 ul of BigDye or ET Terminator reagent
Irrespective of final reaction volume
1/2 = 4 ul BigDye Mix
1/4 = 2 ul
1/8th = 1 ul
Composition of a 1/8th reaction in 5 ul:
BigDye mix 1 ul
5 pmoles primer 1 ul
5X bulking buffer* 0.5 ul
100 ng plasmid template 2 ul
Milli-Q water q.s.p.
Total reaction volume 5 ul
*5X bulking buffer- 400 mM TrisHCl, pH 9.0; 10 mM MgCl2
nota bene: BigDye mix, primer, 5X bulking buffer and water are typically mixed together (i.e., “cocktail”) in a larger volume, and then 3 ul of the cocktail is dispensed to all 384 wells of the thermal cycling plate before adding template. This alleviates the need for dispensing sub-microliter quantities.
We rely on the convection of thermal cycling to mix the template with the cocktail, rather than aspirating to mix, and thereby risking the introduction of bubbles or splattering.
|When using Montage SEQ96 Sequencing Reaction Cleanup Kit, why should I resuspend in Injection Solution vs. Water?||We recommend injection solution for electrokinetic injection. Water does generate higher signal strength for miniaturized reactions, but it is also more difficult to control the electrokinetic injection from water, particularly using MegaBACE. Please also see considerations for injection parameters under "When using Montage SEQ96 Sequencing Reaction Cleanup Kit, what are the optimal injection parameters?".
Injection solution attenuates the electrokinetic injection on MegaBACE and results in more even migration of the sequencing products. Additionally, injection solution widens the range of acceptable template concentration for DNA sequencing, both with the ABI 3700 and the MegaBACE 1000.
We do not suggest resuspension in formamide. If you are using a slab gel, the samples should be resuspended in water, transferred into a separate thermal cycling plate, and vacuum-dried before resuspension in formamide loading buffer. For use with the 3100 and 3100 Avant, resuspend the samples in 25uL injection solution, transfer to the injection plate, and add 50% (v/v) of formamide. Cover plate with rubber mat and put onto sequencer.
|When using Montage SEQ96 Sequencing Reaction Cleanup Kit, what causes loss of short sequencing products? How do I prevent it?||Extended over-drying (2-3 min) of the Montage SEQ membrane during vacuum filtration or processing can cause the loss of short sequencing products. Tailing of C peaks (BigDye v. 3.0 chemistry) or tailing of G peaks (BigDye v. 1.0 and 2.0 chemistry) is further evidence of overdrying. The wells should go visibly empty during vacuum filtration, and optimally, filtration should proceed 30-60 seconds after the fluid in the last well disappears. Blotting of the underside of the plate should be brief and light. The plates should not be left standing on absorbent materials during any stage of processing, since this can cause liquid to wick through the membrane support material on the underside of the plate, and excessively over-dry the samples.
Extensive washing can also cause loss of short sequencing products. Using injection solution for washing and resuspension also appears to be better than water for recovering short sequencing products.
|How do you control evaporation when using Montage SEQ96 Sequencing Reaction Cleanup Kit?||When thermal cycling in volumes less than 5 ul, evaporation can become a problem. We have found that 384 well thermal cycling plates are considerably better than 96 well thermal cycling plates for cycling small reactions. After cycling, we dilute with injection solution prior to transferring to the Montage SEQ plate. This ensures complete volumetric transfer of the small sequencing reaction volumes.|
|When using Montage SEQ96 Sequencing Reaction Cleanup Kit, my signal strength is weak. Why?||Inadequate resuspension and overdrying are the primary reasons for weak signal.
There are 3 options for resuspension of sequencing products:
1. Pipetting resuspension - Vigorous pipetting up and down is important with this method. See note below.
2 Shaking resuspension - Shake vigorously for 10 min on a plate shaker.
3. Passive resuspension - After dispensing injection solution for resuspension into each well, sequencing product will diffuse passively off the surface within 45 min at room temperature.
Pipetting resuspension- Please first verify that the plane of the tips is parallel with the plane of the deck. If you are resuspending by pipetting, adjust your pipettor just above the surface of the membrane. During set-up, this can be accomplished by stepping the pipetting head in small increments toward the membrane surface and then attempting to gently lift the SEQ plate. If the pipette tips are touching the membrane uniformly you will be unable to lift any of the corners or sides of the SEQ plates. Once the plate is unmovable, raise the pipetting head slightly, and then confirm the plate is vertically movable. The pipette tips should be positioned approximately 1 mm from the membrane surface.
A secondary, but less frequent of low signal cause can be inadequate template, this appears to be more of an issue with the BigDye v.3.0 chemistry which was evidently re-formulated to generate a greater proportion of longer sequencing products. Increasing the amount of template added by 1.5 to 2-fold can also increase the proportion of shorter sequencing products.
|Montage SEQ<sub>96 </sub>Sequencing Reaction Cleanup Kit|
|MultiScreen 384 SEQ Plates|