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
Are IPTG and X-gal necessary for blue/white screening of colonies?
With a vector containing the lac promoter and the LacZ alpha fragment (for alpha-complementation), blue/white screening can be used as a tool to select for presence of an insert. X-gal is added to the plate as a substrate for the LacZ-encoded beta-galactosidase enzyme and must always be present to see the blue color in the blue/white screening.
In cases where the lacIq repressor is present (either provided by the host cells on an F' episome, i.e. TOP10F', or expressed from the plasmid), it will repress expression of LacZ from the lac promoter and prevent the formation of the blue color with X-gal. This repression can be reversed by adding IPTG to the media in addition to X-gal, which will inhibit the action of lacIq and re-activate expression from the lac promoter.
Answer Id: 3322
What are some of the competent cell strains offered with your TA Cloning® and TOPO® TA Cloning® vectors, and how should I choose?
TOP10, TOP10F', DH5a™ and INValphaF' are some of the strains offered with the Original TA Cloning® Kits and the TOPO® TA Cloning Kits. E. coli strains DH5a™,TOP10, and INValphaF' do not have the lacIq repressor and permit constitutive expression from the lac promoter. With these cells, there is no need to add IPTG (inducer) for blue/white screening with X-gal. In contrast, E. coli strain TOP10F' carries the lacIq repressor and requires IPTG inducer to be added to enable blue/white screening. Please note, the F' episome in the INValphaF' strain is different from other strains in that it does not contain the lacIq repressor. Usually, presence of the F' and lacIq is only an advantage if you work with an insert that is potentially toxic to the host cell. If your insert is (potentially) toxic, we recommend using the TOP10F' cells without adding any IPTG. The lacIq repressor will repress expression from the lac promoter and you won't get blue-white screening, but you will still get colonies. This way you can clone a toxic construct.
TOPO® TA cloning® kits are also offered with Mach1™ T1r competent cells. The Mach1™ T1 Phage-Resistant (T1R) E. coli strain is the fastest growing chemically competent strain currently available. Doubling time is approximately 50 minutes, compared with an excess of 74 minutes for other cloning strains. Mach1™ colonies are clearly visible within eight hours of plating the transformation mix, enabling you to plate and pick colonies in the same day. With these cells, there is no need to add IPTG (inducer) for blue/white screening.
Answer Id: 3329
Are the TA or TOPO® TA Cloning® vectors available to purchase without competent cells? What about your Zero Background™, Zero Blunt®, or Zero Blunt® TOPO® Cloning Kits
Most of our cloning vectors are offered in a complete format, which includes competent cells. While in most cases other cells can be used with our vectors, we cannot guarantee the results you will get with our cloning vectors if you use your own competent cells. For this reason, most TOPO® TA Cloning® vectors can only purchased in a complete kit with cells, although there are a few exceptions. Non-TOPO® vectors are generally available in multiple formats, with and without cells. To get the most current information on available products, visit the Cloning section of our website under Products & Service.
Zero Background™ and Zero Blunt® vectors are available without competent cells provided, but you should especially careful in choosing competent cells to use with them. These vectors contain the ccdB gene for efficient negative selection of clones without insert, and some E. coli strains are not compatible with the mechanism of negative selection by the lethal activity of the ccdB gene product. In particular, cells with the F' episome have a ccd locus containing the ccdA gene, which prevents ccdB protein cell-killing. Therefore, cells without the F' episome are recommended so that only the CcdB protein will be expressed, and its cell-killing ability will not be inhibited or reduced by CcdA.
Answer Id: 3331
Do PCR products produced with Stoffel fragment have sufficient A-overhangs to work with TA Cloning® kit?
Yes. Stoffel fragment is a 61 kDa modified form of AmpliTaq® DNA Polymerase that lacks 5' to 3' exonuclease activity. It is more thermostable than AmpliTaq® DNA Polymerase, giving it improved performance at the higher denaturating temperatures used with templates that have secondary structure. It more active over a wider range of magnesium concentrations (2 mM to 10 mM MgCl2) than Taq polymerase.
Answer Id: 3332
Can I use a proofreading enzyme such as Platinum®, Pfx™, Vent, or Pfu with TOPO® TA Cloning® kit?
Proofreading enzymes possess 3'-5' exonuclease activity (Gene 112:29 (1992)) which removes 3'-A overhangs necessary for TA and TOPO® TA Cloning® kit. Since the PCR products are mostly blunt-ended, the use of these PCR products in TA cloning yields very low cloning efficiencies. We have developed a simple protocol for adding the 3' A overhang to these PCR products so that they can be used in the TA cloning reaction.
Before starting, you will need the following items:
-A heat block equilibrated to 72 degrees C
-3M sodium acetate
-After amplification with Vent, Pfx, or other proofreading polymerases, place samples on ice and add 0.7-1 unit of Taq polymerase per tube. Mix well. It is not necessary to change the buffer or remove the proofreading polymerase. A sufficient number of PCR products will retain the 3'-A overhangs.
-Incubate at 72 degrees C for 8-10 min (do not cycle).
-Place on ice and use immediately in the cloning reaction.
-If you need to store the samples overnight, extract the sample immediately with an equal volume of phenol:chloroform. Extraction with phenol-chloroform removes all of the polymerase. Precipitate the DNA by adding 1/10 volume of 3M sodium acetate and 2X volume of 100% ethanol. Keep at -20 degrees C for 20 min or -80 degrees C for 10-15 min. Centrifuge at maximum speed for 5 min at room temperature to pellet the DNA. Remove the ethanol, rinse the pellet with 80% ethanol, and allow to air dry. Resuspend the pellet in TE buffer to the starting volume of the PCR amplification reaction. The PCR amplification product is now ready for ligation into the TA cloning or TOPO® TA Cloning® vector.
Note: if your amplification has produced more than one PCR product, you may wish to gel-purify the correct fragment after amplification with Pfu or Vent. After purification, add Taq polymerase buffer, dATP, and 0.5 unit of Taq polymerase and incubate 10-15 min at 72 degrees C. Proceed directly to the cloning reaction.
Answer Id: 3333
Why is there a variety of color and size in the colonies from my plated transformation reaction? How should I optimize screening to have the best chance of finding my clone?
On a typical cloning plate with blue/white screening, there are a few white colonies which do not contain insert. These colonies are usually larger than the other colonies and are due to a deletion of a portion of the plasmid sequence by a rare recombination event (usually from the polylinker to a site in the F1 origin). For the best chance to find a colony with a proper insert, pick clones of a variety of color and pattern for analysis. An insert may generate distinct patterns according to its orientation and size. Even dark blue colonies can have inserts present, if the insert is small and a resulting ORF is in frame with the LacZalpha.
Answer Id: 3334
What polymerases can be used to make PCR products with 3'-A overhangs that are compatible with Invitrogen™ TA Cloning® vectors?
Here is a list of some commonly known PCR enzymes and indication of their compatibility with TA Cloning® vectors.
1) Enzymes that leave abundant 3'-A overhangs, directly compatible with TA Cloning®:
- Invitrogen™ Taq Polymerase, Platinum® Taq Polymerase, Platinum® PCR Super Mix
- Applied Biosystems® AmpliTaq®, AmpliTaq Gold®, and AmpliTaq® 360 DNA Polymerase
- Ambion SuperTaq™ and SuperTaq™ Plus
- Roche Taq Polymerase and Tth Polymerase
- TaKaRa Taq
- Agilent Taq2000 polymerase, Exo-Pfu Polymerase
2) Enzymes that leave partial A overhangs, generally contain mix of Taq and proofreader and may have lower efficiency with TA Cloning®:
- Invitrogen™ Platinum® Taq High Fidelity and Platinum® PCR Supermix High Fidelity
- Roche Expand System
- TaKaRa LA Taq and Ex Taq
Answer Id: 3335
Can a phosphorylated PCR product be TA-cloned?
Phosphorylated products can be TA-cloned, but not TOPO®-cloned. This is because the necessary phosphate group is already contained within the Topoisomerase-DNA intermediate complex on the TOPO® vectors. The TOPO® plasmid has a 3' phosphate, to which Topoisomerase is covalently bound. The PCR product must provide 5'OH and 3'OH groups to react with the Topoisomerase, and presence of an additional phosphate group will inhibit the Topoisomerase-mediated ligation.
The non-TOPO® TA Cloning® vectors have a 3'OH and a 5' phosphate, and because they involve use of standard ligase, they are compatible with PCR products both with or without 5' and 3' phosphate groups.
Pre-phosphorylated PCR products can be phosphatase-treated (CIP/BIP) to remove the phosphate groups before TOPO® cloning - however, cloning efficiency may be decreased compared with an insert that was never phosphorylated at all.
Answer Id: 3336
How does the pCR®4-TOPO® and pCR®4Blunt-TOPO® streamline sequencing?
Both the pCR®4-TOPO® and pCR®4Blunt-TOPO® vectors contain a minimized multiple cloning site. There are only 33 base pairs from the sequencing primer sites to the cloning site. This allows for more sequencing read of your insert and less of the vector which an save time analyzing the sequence data.
Answer Id: 3337
Is it possible to generate nested deletions using the TOPO® Cloning Kit for Sequencing and the ZeroBlunt TOPO® PCR Cloning Kits?
Yes. Both vectors (pCR®4 and pCR®-Blunt II) are designed to allow the creation of nested deletions for sequencing large inserts using the same sequencing primer. In addition, a comprehensive protocol is provided in the TOPO® manual for these kits.
Answer Id: 3338
Why is there a TOPO® Cloning kit "Stop Solution" mentioned in my manual, but it is not supplied in the TOPO® Cloning kit?
Stop Solution is a reagent included in older versions of all TOPO® kits, and still offered in the TOPO® XL kits. It is very similar to the current TOPO® cloning kit Salt Solution. Instruction manual versions preceding Version J recommended adding the Stop Solution at the end of the 5 minute TOPO® reaction. The basic function of the Stop/Salt Solution is to keep the Topoisomerase from re-binding to the plasmid DNA and nicking the DNA. Later studies found that it was actually more effective to add this component during the TOPO® cloning reaction versus afterward, and thus the Stop Solution was replaced with the similar but higher-concentration Salt Solution in most kits.
Answer Id: 3339