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查看更多产品信息 CloneChecker™ System (for screening bacterial colonies) - FAQs (11666013)
11 个常见问题解答
Depending on the copy number of the plasmid, the number of cells collected, and their stage of growth, it is estimated that ~2 to 20 ng are obtained for analysis. This amount is suitable for one gel analysis. Typically, the Supercoiled Analysis method recovers more plasmid than the Restriction Enzyme Analysis method. Please note, plasmid is not purified with this system, but rather only released from cells in manner suitable for characterization. Plasmids remain contaminated with most of the cell constituents.
Restriction Enzyme Analysis and Supercoiled Analysis are the methods provided in the CloneChecker system. Restriction Enzyme Analysis is useful for positively identifying clones from a characteristic digestion pattern. Insert size and orientation can be determined reasonably accurately by choosing the appropriate restriction endonuclease(s) and an appropriate DNA molecular size marker. This screening procedure makes sense when the probability of finding the correct clone within the population of colonies on a plate is reasonably high, or when confirmation of a clone resulting from a preliminary screening is desired. Supercoiled Analysis is useful when the recovery frequency of an insert-containing vector is low or when the desired insert is within a certain size range. Within a few minutes of picking a clone, a sample can be loaded on an agarose gel for determination of supercoiled size without the extra time or expense of performing a restriction digest on the plasmids. Both methods avoid overnight culture of each clone and the work plasmid DNA minipreps for this type of analysis.
The system contains sets of proprietary chemical solutions that release nucleic acids, especially plasmids, from bacterial cells. Typically, released plasmids are then characterized physically by supercoiled size or by restriction digestion pattern using agarose gel electrophoresis.
Plasmids ranging in size from 2.7 kb to 13 kb have been analyzed, and the CloneChecker System has been used to isolate a 46 kb cosmid for quick analysis. It should be possible to assay for larger DNAs, such as cosmids, providing a sufficient amount of DNA is detectable.
Restriction endonuclease digestion of released plasmid DNA was successful with 20 of the popular enzymes used in cloning: Acc I, Ava I, BamH I, BstE II, Bgl II, Cla I, EcoR I, Hpa I, Hind III, Kpn I, Nco I, Nhe I, Not I, Sac II, Sal I, Ssp I, Sst I, Spe I, Xba I, and Xho I. In addition to single digestions with these enzymes, a number of double digests were successful as well. No enzyme tested has failed to perform properly. Note, restriction digests are only possible with plasmid released with the GREEN solution from the Restriction Enzyme Digest method.
Plasmids were successfully analyzed from E. coli DH10B, DH5 alpha, HB101, STBL2, XL-1 Blue, JM109, NM522, and Top10' cells. Yield of the same plasmid can vary between strains depending upon individual growth characteristics. For example, STBL2, a slower-growing plasmid gives a reduced plasmid yield.
Clones may be recovered in a number of ways after using the CloneChecker System:
(1) The initial cell suspension in growth media uses only one-half of the volume for processing, leaving 3 µL for inoculation of new media or a secondary agar plate.
(2) Cells are incompletely transferred from the agar plate, leaving many cells to regrow the colony when the plate is returned to the incubator.
(3) A second agar plate marked with an identifying grid is inoculated by the tip after initial transfer of cells to the 6 µL of media.
The CloneChecker System can be used to analyze plasmid DNA from different strains of E. coli grown on an agar plate, in liquid media, or stored as a glycerol stock.
The supercoiled analysis method offered by the CloneChecker system is an alternative to PCR for screening recombinant bacterial colonies or liquid cultures. Supercoiled analysis is significantly faster and less expensive than colony PCR for screening large numbers of clones. When the cloning efficiency is low, the savings in labor, time and reagent expense using the CloneChecker system can be significant. In standard PCR, reaction efficiency drops dramatically for amplicons greater than 1 kb and insert orientation is not usually determined. These limitations are overcome using the CloneChecker system.
The CloneChecker System is a rapid method for screening recombinant bacterial colonies or liquid cultures of colonies for the presence of target plasmid DNA. The CloneChecker System:
- Screens bacterial colonies for plasmid DNA and insert of interest prior to purification
- Screens for inserts >200 bp in plasmids up to 15 kb by size comparison to the original vector or a DNA size marker
- Goes from plated colonies through lysis in less than 5 minutes
- Allows for supercoiled, restriction digest, or PCR analysis
- Works with single-copy plasmids starting with a 2-mm bacterial colony
- Includes sufficient reagents for processing 100 bacterial samples by supercoiled analysis (direct size comparison); and 100 samples by restriction endonuclease or PCR analysis.
The amplified DNA needs to be purified from the PCR mixture components prior to cloning. The dNTPs carried over from the PCR are competitive inhibitors for ATP in the ligation reaction.
If during synthesis of the PCR primers their chemical integrity has been compromised by either a base substitution or modification, the enzyme recognition site may in actuality not exist. If this is the case, PCR products will be resistant to digestion with restriction enzymes. It may be necessary to use a higher concentration of the restriction enzyme and to incubate at the appropriate temperature overnight to ensure cutting.