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Technical Service
If you have technical questions about DeWalch Life Technologies products or services, or any molecular biology procedure associated with a DeWalch Life technologies product, contact our technical service at (800) 880-8993 ext. 247 Monday through Friday during the hours 8:30 A.M. and 5:00 P.M. or e-mail.
Specific Questions Which Came from Customers Previously
1. Can I isolate plasmid DNA from gram-positive bacteria (such as Streptomyeces)?
The protocol (no centrifugation) and solutions included are not suitable to lyse gram-positive bacteria such as Streptomyces. However, some of our clients have combined their own successful protocols with our kits for the plasmid purification.
In these cases a crude DNA preparation is obtained by lysis of the bacterial cell pellet (but not bacterial liquid culture) by a user-specific lysis method, usually lysis of the cells by a lysatic enzyme and then by our solutions, and subsequently precipitation of the nucleic acid (both genomic and plasmid DNA) with isopropanol, along with our lysis/binding solution. The resulting plasmid DNA is resuspended and eluted, while majority of the genomic DNA is hold on the surface of each well. The rest of the procedure follows the standard protocol.
2. I want to isolate a low-copy plasmid. The amount of plasmid DNA can be bound on a DeWalch plate, but the culture volume exceeds the suggested values.
For the isolation and purification of low-copy plasmid from large volume (up to 1.8 ml per well), we recommend our Ultra Pure Plasmid Purification Kits. However, if user really wants to use the sequencing-grad plasmid DNA purification kits, we suggest to centrifuge down cells and then, lysis the bacterial cells by adding the solution I, and following the rest of the procedure. However, the disadvantages are threefold. First, because of higher volumes, or higher numbers of bacterial cells, longer lysis time is required. Second, if un-lysed bacterial cells are presented, some wells of the plate might clog. Finally, if such high amounts of RNA are bound to each well, it cannot be guaranteed that all RNA is digested and washed off the well. There could be a small amount of RNA that is carried over into the elute.
We recommend using our Ultra-Pure Plasmid Purification for low-copy number plasmid purification.
3. Can I isolate and purify chromosomal/genomic DNA with the sequencing-grad plasmid DNA purification kits?
Isolation and purification of DNA with a size of >40 kb is possible with the kits, but requires a modification. However, for preparation of such high molecular weight DNAs we strongly recommending our genomic DNA purification kits.
4. Can I isolate large plasmids, cosmids, or BAC DNA with the sequencing-grad plasmid purification kits?
Plasmids with sizes up to 40 kb are well-suited to be purified with DeWalch Sequencing-Grade Plasmid Purification Kits. However, large plasmid DNA as cosmids or BACs are usually present as low or single-copy plasmid per cell. Thus, higher culture volumes are needed to produce a reasonable DNA yield. Furthermore, a gentle sample handling procedure is required in order to avoid break-up larger plasmids, cosmids or BACs. We recommend using our Ultra-Pure Plasmid Purification Kits for isolation of large plasmids, cosmids, or BAC.
5. Is it possible to isolate ?-DNA or M13 DNA with DeWalch Sequencing-Grad Plasmid DNA Purification Kits?
It is possible to isolate ?-DNA or M13 DNA with DeWalch Sequencing-Grad Plasmid DNA Purification Kits. However, modified protocols are needed. Protocols are available upon request.
6. Can I reduce the elution volume?
To achieve a better DNA yield and concentration, we recommend using a minimum of 100 µl of elution buffer. Higher DNA yield and concentration can be improved by using warm (60-65ºC) elution buffer and/or incubating the plate for at least 5 min. at 37ºC with a gentle agitation after pipetting the elution buffer onto the silica membrane. A further reduction of the elution volume will not further increase the DNA concentration but will only the increase total DNA yield.
7. Can I use the Sequencing-Grade DNA Purification kits prepared DNA for transfection?
We don’t recommend the Sequencing-Grade plasmid for transfection since the plasmid still contains a significant amount of bacterial endotoxins and chromosomal DNA that can affect a subsequent transfection. For transfection, we recommend using DeWalch’s Ultra Pure Plasmid Purification Kits.
DeWalch Sequencing-Grade NeXprep Plasmid Purification Kit Extended Trouble Shooting Guide
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Problem Symptoms |
Possible Causes and Corrective Actions |
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Filter/Drip Clogging |
Breaking the filter membrane during inoculation allowing binding media to clog the drip directors. Make sure not to disturb the binding media and filter during inoculation process. Bottom of the well should appear white after processing.
Centrifugation force too high to force the binding matrix to clog the drip director. Centrifuge the plate for 5 min. at <1,400 xg.
Low or inconsistent vacuum. Check pump, traps and ensure valves are open. |
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Slow draining and drying |
Low vacuum. Increase vacuum, free constricted tubing (do not use thin wall tubing).
Cell pellets too old. Use fresh-grown cells if possible. Store cell pellets (plate) for a few hours on ice, or frozen at -20ºC or -80ºC for a short period (up to 2 weeks). |
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Low yields overall |
Only high-copy plasmids should be prepared with the Sequencing-Grad plasmid DNA purification Kits in order to have acceptable plasmid yields.
Insufficient agitation of cultures during incubation, recommend agitating at 340 – 420 rpm.
Old or weak bacterial inoculation. Use fresh transformants of remake glycerol stocks for inoculation.
Growth medium. Use richer media such as 2X YT or
TB as growth medium.
Over-drying of the silica matrix. Perform the step 8 (continue applying vacuum) for a appropriate period of time, and check the plate dryness periodically by patting the plate on a paper towel and followed recommended vacuum period of time according to the vacuum pressure
(Table 1, page 13, the user manual). |
|
Spotty Yields |
Bacterial inoculums are too old. Use fresh bacterial colonies or glycerol stocks for inoculation. |
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Degraded plasmid DNA |
Elution with sterile water. Elution with sterile water is possible, but in some circumstance DNA eluted in water is less stable than in a Tris-buffered elution buffer.
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The E. coli strain is known as problematic. Certain bacterial strains contain high amounts of endo- and exonucleases. Lower growth temperature (~30ºC), harvest bacteria immediately and continue plasmid isolation.
Over grown bacterial cultures. Do not over grow bacterial cultures. For an ampicillin-resistance bacterial strain, do not grow for longer than 16 hrs. |
|
RNA contamination |
If the bacterial culture grows to much higher cell densities than 1 x 109 cells per ml the plate may be overloaded. It can be resulted from using a very rich medium and higher inoculums. In such as case the lysis procedure and digestion of the cellular RNA (with RNase) are incomplete since the buffer volumes are insufficient to process such large quantities of cells. Avoid over-grow and/or over-inoculate the bacterial culture.
Insufficient RNase digestion time. Make sure incubate for 2 min at room temperature after adding Buffer B (step 3, the manual), and incubate the DNA samples for 30 min at room temperature if residual RNA is still seen on the gel.
Has a very “dirty” E. coli strain been used? Some E. coli strain produces extreme amounts of RNA. In such circumstance, add fresh RNase to Buffer B to a final concentration of 200 µg/ml.
RNase has lost activity. We have found enough RNase activity to digest all the cellular RNA even after storage of the Buffer B for 6 months at room temperature. However, we recommend adding fresh RNase to a final concentration of 100 µg/ml to ensure the RNase full activity. ” |
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Low or No Sequencing Signal Strength |
Excess alcohol in the final elute. In some case, the eluted DNA smell quite strongly of alcohol and/or DNA sample float out of the well of an agarose gel. Subsequently, DNA sequencing reactions will be inhibited. We recommend to get rid of the alcohol by incubating the plate (without seal) for 10 min. at elevated temperatures (i. e., 50ºC), or by an alcohol precipitation and resuspend the DNA pellet in Tris buffer.
Improper amount of template DNA. In our study, a significant improvement of reading length (QV20 LOR), from 425.8 to 655.1, has been revealed when increasing the volume of template from 1 ul to 3 ul (approximately 150ng of plasmid DNA). |
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Dirty Sequencing |
The trace peaks are misshapen and/or contain secondary peaks under the primary peak. The total quality scores counts are generally low, and the sequencing peaks are very weak. In such case, contamination of RNA or low amount of DNA template used might be the cause. We recommend running an agarose gel or checking 260/280 ratio to verify purity and concentration of the DNA sample.
Further incubation of the plasmid sample (for 30 min) at room temperature to get rid of the cellular RNA, or cleaning and/concentrate the DNA by alcohol precipitation if needed.
The sequencing trace has two or more sequence peaks at the same location. The secondary peaks may be the same height as the primary peaks down to about 20%.
The quality scores are low with often less than 100 Q20+ bases. It could be caused by mixed clone(s) of the culture. We recommend resolving library problems and preparing new DNA templates. |