Template Preparation for Automated DNA Sequencing..
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Our lab routinely produces high quality data with read lengths to 700 bases; however, this depends on many factors,
including how clean the template DNA is, or the annealing efficiency of the primer. To prepare DNA which is clean enough for automated
sequencing, please take a careful look at this page!
Culture Conditions
Preparation methods
Preparation Kits recommended
Clean-up of DNA prepared by "old-fashioned" methods
Clean-up of PCR products
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- Culture Conditions:
- Do not overgrow E. coli cells. Overgrowth can result in release of chromosomal DNA and polysaccharides that
co-purify with plasmids and inhibit sequencing reactions. We have been told that richer media, especially buffered ones, delay this
process
- A 0.5 M NaCl wash of the cells prior to lysis can be very helpful in removing polysaccharides and media
components that often copurify with the DNA and inhibit sequencing reactions. Simply resuspend the cell pellet completely by
vortexing, then re-spin.
- Preparation methods:
- Plasmid templates can be prepared using a variety of protocols. We have found that purification columns designed
specifically for Automated Sequencing work best (see below). However with a certain amount of trouble-shooting standard alkaline lysis,
boiling, or CsCl methods are acceptable. The cleaner the DNA, the better the sequence!
Miniprep kits do not ensure cycle-sequencing grade DNA!
- Preparation Kits recommended:
- (NOTE: all QIAGEN/miniprep products are not equal)
- QIAwell Plus/UltraPlasmid Kit (cat #16142, 16144)
- QIAwell Plasmid purification system (cat#171220, 17124)
- QIAGEN Plasmid Mini kits (cat# 12123 or 12125)
- Promega Wizard Plus SV minipreps (cat#A1340)
- ABI PRISM Miniprep kit (cat #402789)
- Clean-up of DNA prepared by "old-fashioned" methods:
- For standard alkaline or boiling minipreps, it is important to avoid the co-precipitation of inhibitors from the
cell lysate, especially when columns are not used for purification.
- Use only the minimal amount of room temperature isopropanol
(do not chill) for precipitation (0.60 volumes). Do not wait to spin and do not spin longer than 5 minutes to precipitate the DNA.
- For many samples, including DNA precipitated from CsCl solutions (dialysis is better), one or more ammonium
acetate precipitations are helpful to purify the DNA. Add 1/2 volume of 7.5 M ammonium acetate, mix well, then add 0.6 volumes
of room temperature isopropanol, mix well, and spin. Residual CsCl in DNA
samples will cause failures!
- The majority of the RNA should be removed from miniprep DNA, by using RNAse, high salt precipitation or column
purification.
- Clean-up of PCR products
- It is critical that all unextended primers left over after the PCR reaction are removed prior to sequencing. Regular
DNA precipitation methods are not appropriate. You must gel-purify your PCR products, or run them through an appropriate spin column.
- Another alternative is to use a PCR clean-up product such as QIAquick(cat# 28104, 28106).
- We can clean them for you. The Core offers an inexpensive PCR cleanup
for individual tubes or 96 well plates. See Purification of PCR products under Services.
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