How can I tell whether or not a reagent bottle is pressurizing correctly when using the Procise™ System?
You can backflush the bottle's pickup line in manual control (there is a specific function for each bottle position on the Procise™ System) and observe its bubbling, which should slow and then stop within a short period (depending upon how full the bottle is) as it pressurizes with argon. If it continues to bubble, either the cap assembly is leaking or the pressurizing or venting valves for the bottle are.
Answer Id: 1261
Why is the conductivity so high in my peptide synthesis (monitored during deprotection)?
The meter detects any ionic species. A common cause of higher than expected values is a leak of a small amount of resin from the RV into the lines and up to the in-line filters. The use of old or poor quality piperidine or NMP may also give a high background. Standard conductivity measured in micro Siemens/cm is much higher than the sensitivity of this cell. A very small amount of ionic material caused a large change in the reading. Occasionally, Fmoc amino acids have ionic contaminants which give high readings. In-line filters may also be contaminated.
Answer Id: 1313
What is 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.
Answer Id: 3027
How does the CloneChecker™ system compare to colony PCR?
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.
Answer Id: 3074
Which PCR polymerases do you recommend for TA/Blunt/D-TOPO cloning and why?
A. TA Cloning:
- This cloning method was designed for pure Taq polymerases (native, recombinant, hot start); however, High Fidelity or Taq blends generally work well with TA cloning. A 10:1 of 15:1 ratio of Taq to proofreader polymerase will still generate enough 3 A overhangs for TA cloning
- The recommended Life Technologies® polymerases include Platinum® Taq, AccuPrime™ Taq, Platinum® or AccuPrime™ Taq High Fidelity, AmpliTaq®, AmpliTaq Gold®, or AmpliTaq Gold® 360.
B. Blunt cloning:
- Use proofreading enzymes such as Pfx50, Platinum® or AccuPrime™ Pfx.
C. Directional TOPO cloning:
- Pfx50 or AccuPrime Pfx work well.
Answer Id: 6651
Can I use TOPO®TA pCR2.1 or pCR II or pCR4 for my protein expression experiments?
How does blue/white screening work?
If working with a vector that contains the lac promoter and the LacZ alpha fragment (for α complementation), blue/white screening can be used as a tool to select for presence of the insert. X-gal is added to the plate as a substrate for the LacZ enzyme and must always be present for blue/white screening. The minimum insert size needed to completely disrupt the lacZ gene is >400 bp. If the LacIq repressor is present (either provided by the host cells, for example TOP10F, or expressed from the plasmid) it will repress expression from the lac promoter, thus preventing blue/white screening. Hence in the presence of the LacIq repressor, IPTG must be provided to inhibit the LacIq. Inhibition of LacIq permits expression from the lac promoter for blue/white screening. X-gal (also known as 5-bromo-4-chloro-3-indolyl β-D-glucopyranoside) is soluble in DMSO or DMF, and can be stored in solution in the freezer for up to 6 months. Protect the solution from light. Final concentration of X-gal and IPTG in agar plates: Prior to pouring plates, add X-gal to 20 mg/mL and IPTG to 0.1 mM to the medium. When adding directly on the surface of the plate, add 40 μl X-gal (20 mg/mL stock) and 4 μl IPTG (200 mg/mL stock).
Answer Id: 6664
How does ccdB selection work?
TOPO® vectors containing the LacZ-ccdB cassette allow direct selection of recombinants via disruption of the lethal E. coli gene, ccdB. Ligation of a PCR product disrupts expression of the LacZ-ccdB gene fusion permitting growth of only positive recombinants upon transformation. Cells that contain non-recombinant vector are killed upon plating. Therefore, blue/white screening is not required. When doing blue/white color screening of clones in TOPO® vectors containing the LacZ-ccdB cassette, colonies showing different shades of blue may be observed. It is our experience that those colonies that are light blue as well as those that are white generally contain inserts. The light blue is most likely due to some transcription initiation in the presence of the insert for the production of the lacZ alpha without enough ccdB expressed to kill the cells and is insert dependent. To completely interrupt the lacZ gene, inserts must be >400 bp; therefore an insert of 300 bp can produce a light blue colony. A white colony that does not contain an insert is generally due to a spontaneous mutation in the ccdB gene.
A minimum insertion of 150 bp is needed in order to ensure disruption of the ccdB gene and prevent cell death. (Reference: Bernard et al., 1994. Positive-selection vectors using the F plasmid ccdB killer gene. Gene 148: 71-74.) Strains that contain an F plasmid, such as TOP10F, are not recommended for transformation and selection of recombinant clones in any TOPO® vector containing the ccdB gene. The F plasmid encodes the CcdA protein, which acts as an inhibitor of the CcdB gyrase-toxin protein. The ccdB gene is also found in the ccd (control of cell death) locus on the F plasmid. This locus contains two genes, ccdA and ccdB, which encode proteins of 72 and 101 amino acids respectively. The ccd locus participates in stable maintenance of F plasmid by post-segregational killing of cells that do not contain the F plasmid. The CcdB protein is a potent cell-killing protein when the CcdA protein does not inhibit its action.
Answer Id: 6665
How does selection with the LacZ gene work?
If working with a vector that contains the lac promoter and the LacZ α fragment (for α complementation), blue/white screening can be used as a tool to select for presence of the insert. X-gal is added to the plate as a substrate for the LacZ enzyme and must always be present for blue/white screening. The minimum insert size needed to completely disrupt the lacZ gene is >400 bp. If the LacIq repressor is present (either provided by the host cells, for example TOP10F, or expressed from the plasmid), it will repress expression from the lac promoter thus preventing blue/white screening. Hence, in the presence of the LacIq repressor, IPTG must be provided to inhibit the LacIq. Inhibition of LacIq permits expression from the lac promoter for blue/white screening.
Answer Id: 6666
Im getting low to no colonies after transformation. Why?
Some possible causes and remedies are:
- Ligase function is poor. Check the age of the ligase and function of the buffer.
- Competent cells are not transforming. Test the efficiency of the cells with a control supercoiled vector, such as puc19.
- Both molecules were de-phosphorylated.
- Inhibition of ligation by restriction enzymes and residual buffer. Try transformation of uncut vector, clean up restriction with phenol, or carry out PCR cleanup/gel extraction before ligation.
- Incorrect antibiotic selection used. Check the plasmid and plates and make sure concentration of antibiotic used is correct.
If nothing above applies, low to no colonies may be due to instability of the insert DNA in your competent cells. In this case, E. coli strains such as Stbl2™, Stbl3™, or Stbl4™ have been shown to support the propagation of DNA with multiple repeats, retroviral sequences, and DNA with high GC content better than other strains.
Answer Id: 6667
The clones Im selecting show deleted inserts. Why?
This may be caused by the instability of the insert DNA in TOP10 E. Coli. In this case, E.coli strains such as Stbl2™, Stbl3™, or Stbl4™ have been shown to support the propagation of DNA with multiple repeats, retroviral sequences, and DNA with high GC content better than other strains.
Answer Id: 6668
Im able to see colonies on a plate, but when I pick them for liquid culture, no growth is observed. Why?
One possible explanation could be toxicity associated with the insert. This toxicity does not affect slow growing cells on solid medium but is much stronger in faster growth conditions like liquid medium.
1. Use TOP10F or any other strain with the LacIq repressor
2. Try using any other strain appropriate for cloning.
3. Lower growth temperature to 27 - 30 degrees C and grow the culture longer
4. Another possibility to explain lack of growth is possible phage contamination. In this situation we recommend using an E. coli strain that is T1 phage resistant like DH5alpha-T1R.
Answer Id: 6669
I see small satellite colonies on my LB+Amp plates. Why is this?
These small colonies are most likely caused by degradation of the Ampicillin. The colonies are just untransformed cells that grow on LB with degraded Amp. In order to circumvent this scenario, you can try to:
1. Plate cells at a lower density
2. Use fresh LB-Amp plates or replace Ampicillin with carbenicillin.
3. The plates should not be incubated for more than 20 hours at 37 degrees C. Beta-lactamase, the enzyme produced from the Ampicillin-resistance gene, is secreted from the Amp-resistant transformants and inactivates the antibiotic in the area surrounding the transformant colony. This inactivation of the selection agent allows satellite colonies (which are not truly Amp-resistant) to grow. This is also true if carbenicillin is being used.
Answer Id: 6670
Im trying to decide between the TOP10, DH5α™, and Mach1™ strains you have for my TOPO® TA Cloning® reactions. Can you explain the significant differences between these strains?
DH5α™ cells are commonly used for routine cloning, but are mcr/mrr+, and therefore not recommended for genomic cloning. The TOP10 competent cells, on the other hand, contain mutated mcr/mrr, and therefore are a good choice for routine cloning and can be used for cloning of methylated DNA, such as eukaryotic genomic DNA. Our Mach1™ strain is the fastest growing cloning strain that is T1 phage resistant.
Answer Id: 6704
What are some tips you can give me to obtain the highest transformation efficiency?
Some suggestions that will help you to obtain the highest transformation efficiency are:
- Thaw competent cells on ice instead of room temperature; do not vortex cells.
- Add DNA to competent cells once thawed.
- Ensure that the incubation times are followed as outlined in the competent cell protocol for the strain you are working with; changes in the length of time can decrease efficiency.
- Remove salts and other contaminants from your DNA sample; DNA can be purified before transformation using a spin column, or phenol/chloroform extraction and ethanol precipitation can be employed.
Answer Id: 6709