Screening for Recombinants - Promega

More documents

Recommendations

Info

Screening for Recombinants Troubleshooting Subcloning Experiments Symptoms Possible Causes Comments Few or no colonies obtained Cells not competent Competent cells may exhibit lower after transformation transformation efficiencies 5–6 weeks after preparation. To verify that bacteria are competent, perform a test transformation using a known amount of a standard supercoiled plasmid (see page 47). Unsuccessful ligation Analyze samples of a linearized vector and the vector + insert ligation on an 0.8% agarose gel. If ligation was successful, the banding pattern of the ligation products should be different from that of the unligated sample. Inactive T4 DNA Ligase Verify that the T4 DNA Ligase is active; perform a control ligation reaction with linear plasmid DNA. Inactive T4 DNA Ligase Buffer Store T4 DNA Ligase 10X Reaction Buffer in small aliquots at –20°C to minimize freezethaw cycles of the buffer. Multiple freezethaw cycles may degrade the ATP in the buffer. Digested vector ends are not Restriction enzyme sites that are adjacent compatible with the fragment within the multiple cloning region or near the ends can prove difficult to digest completely. See page 40 for a method to create PCR products with restriction sites in the primers. Excess ligation products The added ligation products should not added to competent cells exceed 0.5% of the transformation reaction volume. Excess DNA (>10ng) may also inhibit the transformation. High Background Unsuccessful dephosphorylation Attempt to religate the dephosphorylated of vector DNA vector. It should religate with low efficiency. Plates lack the correct antibiotic; Perform a mock transformation with no DNA the antibiotic is inactive added. If colonies grow, discard the plates. Ratio of linearized, phosphorylated Reduce the amount of linearized vector in vector to insert DNA is too high the reaction. Religation of the vector is favored when the vector:insert ratio is too high. 54 Promega Subcloning Notebook
Screening for Recombinants Troubleshooting Subcloning Experiments—Deletions of the Insert During screening of your recombinant plasmids, you may encounter a situation where part of your insert is deleted. Perhaps careful examination of the subcloning strategy has not identified steps that may have led to this deletion. You attempt to sequence these clones and find that a portion of the insert has been deleted along with part of the vector. How did this happen? Two possibilities come to mind that could lead to such an event. Symptoms Possible Causes Comments Recombinants are isolated Insert is unstable in the host strain The insert may have been a substrate for recombut contain deletions (usually bination by recombinases in the most bacterium unidirectional deletions of (remember most common laboratory strains are insert and part of vector) recA minus, but there are other recombinases present). You can transform the plasmid into an E. coli strain deficient in more recombinases than just the recA. Some strains like SURE ® cells from Stratagene are deficient in recombinases recB and recJ and may allow you to propagate the unstable insert. Also try growing at a lower temperature. Insert is toxic to the host strain Certain inserts may produce toxic gene products. The bacteria responds by deleting a portion of either the plasmid, the insert or both. High copynumber plasmids will tend to produce more toxin and thus be more prone to deletion or rearrangement. Most cloning plasmids carry the modified ColE1 origin of replication derived from pUC vectors, which maintain the copy number of the plasmid as high as 100–400 copies per bacterium. Low-level, leaky transcription in this high copy number plasmid can yield significant quantities of the toxic product. One solution is to transfer the insert to a different vector with a lower copy number. Promega has some vectors with lower copy numbers available like the pALTER ® -1 (based on the pBR322 ColE1 origin; as few as 25 copies per cell) and the pALTER ® -Ex2 (based on the pACYC origin of replication; ~10 copies per cell). This solution requires moving to a new vector. If you need to stay with the same vector (e.g., a mammalian expression vector) there are E. coli strains that have mutations that limit the copy number of pUC-based ColE1 origin of replication. The ABLE ® strains from Stratagene will reduce copy number four- to ten-fold compared to more common laboratory strains. www.promega.com • techserv@promega.com 55
Page 1 and 2: Screening for Recombinants Introduc
Page 3 and 4: Screening for Recombinants Go Direc
Page 5: Screening for Recombinants Plasmid

Screening for Recombinants - Promega

You also want an ePaper? Increase the reach of your titles

Delete template?

Save as template?