When isolating P1 constructs and other very low copy number plasmids using the Nucleobond AX alkaline lysis-based protocol, the potential always exists for lower than expected yields to be obtained. While, in general, there are a number of factors that could adversely affect plasmid yield (most of which are addressed elsewhere in this guide), in the case of low copy number plasmids, incomplete bacterial lysis is the number one culprit. The primary reason for this is that in order to get the maximum yield for a particular cartridge size, many researchers grow cultures of these plasmids that are larger than can be efficiently handled by the prescribed volumes of lysis buffers for the cartridge. The first and most obvious solution is to increase the amounts of the lysis buffers to be used in these cases to reduce viscosity and to promote diffusion. However, how much is sufficient for a particular culture? Are there any other ways to alleviate the incomplete lysis problem?
(A) Some Golden Rules for Lysis Buffer Volumes
Whether the copy number of your plasmid is high or low, there are certain simple rules regarding the minimum volumes of the lysis buffers, S1, S2, and S3, to be used. These are as follows:
Use a minimum of 1.0 ml of each lysis buffer per 100 ml of culture regardless of which cartridge size is being used. As in (1) above, if the particular cartridge requires more than this then use the prescribed amount for the cartridge. For example, if a 100 ml CAPped culture of a low copy plasmid is to be processed on an AX-500 cartridge then use the prescribed 12 ml of S1, S2, and S3 for the cartridge. On the other hand, if a 500 ml CAPped culture of a low copy number is to be processed on an AX-500 cartridge then one would still use the prescribed 12 ml of S1, S2, and S3 for the cartridge according to this rule to insure proper lysis.
AX 20 - 3 mL of culture
AX 100 - 20 mL of culture
Years ago before the advent of today's extremely high copy number plasmids, cultures were routinely CAPped with an antibiotic to which the bacteria with plasmid were not resistant. CAPping shuts down the protein production machinery of the cells, but permits the plasmid DNA to continue to replicate, thus increasing its copy number. CAPping is not done with high copy number plasmids because it is not needed and probably would have little if any effect, anyway.
Chloramphenicol dissolved in ethanol is probably the most often used antibiotic for CAPping usually in the concentration of 150-170 µg/ml of culture. Other antibiotics like spectinomysin are used when the plasmid confers chloramphenicol resistance.
Induction is done on cultures of P1 plasmids with the same purpose as CAPping, that is to increase copy number. While similar in concept at least to CAPping, it does not require antibiotics. Instead, treating a P1 culture with IPTG can effectively increase copy number from 1 to about 20. Unlike CAPping, P1 cultures that have been induced should still be handled following rule 'A' above.
In cases were CAPping or induction is planned on being used, smaller starting culture volumes can be used to generate the same amount of plasmid as larger non-CAPped or non-induced ones. Thus, smaller amounts of the S1, S2, and S3 lysis buffers could be used, although for very large DNA (BACs and PACs) use of a more dilute solution is preferred to assist diffusion from the cell.
(C)Use of an additional volume of N5 Elution Buffer
If the elution step is repeated one additional time (see Step 8, in A Modified Alkaline Lysis Procedure for the Purification of Plasmids and Cosmids), up to 30% more DNA can be isolated. This is especially true for the AX-100 and AX-500 cartridges. Note the flow rates of Nucleobond AX cartridges are up to two times faster than for Qiagen Tip cartridges which means that clogging from higher viscosity solutions is less likely (less sensitive to cell density problems), and the amount of losses from endonucleases will be lower due to shorter contact times.
(D)Use of a Denaturing Elution Buffer
It has been reported in some cases that the use of a high GC content type of elution buffer helps to increase the yields of very large plasmids such as P1 constructs and BACs (bacterial artificial chromosomes) regardless of their GC content. The formula for this elution buffer is as follows: 50% formamide, 1.0 M KCl, 15% EtOH & 0.1 Tris-phosphate, pH 8.5. This buffer should be heated to 60C before loading. For the recommended procedure for preparing this buffer refer to N5 and L5 Elution Buffer Preparation. However, use of this formamide buffer can lead to complications in the precipitation step unless care is taken to prevent salt precipitation (room temperature propanol precipitation and centrifugation is required), formamide removal (a second propanol precipitation is required) and propanol removal (an additional ethanol precipitation is required).
For further suggestions please see A Modified Alkaline Lysis Procedure for the Purification of Plasmids and Cosmids.
Having specific problems? Please refer to the Nucleobond AX Trouble Shooting Guide.
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