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Product FAQ

What can be done when I observe errors on the 433A such as "chemistry not declared" (CND), a "PAUSE" that will not unPAUSE, a "run cannot be sent", or during a send the chirps are slower than usual?

Answer

The problem occurs when a 433A connected to a Macintosh™ computer interprets a signal from the computer as a "reset". Some "fixes" have been successful, particularly installation of a special optical isolator on the 433A. When the instrument has frozen in a run, the run needs to be stopped and re-started, often with a "COLD REBOOT". To prevent the problem, it is strongly suggested that no network connection be attached to the computer during a run. No other software should be run, or even loaded to the hard drive of the computer.

Answer Id: E1312

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Product FAQ

Why is it necessary to dilute ligated DNA products before adding them to competent bacterial cells?

Answer

Components of the ligation reaction (enzymes, salts) can interfere with transformation, and may reduce the number of recombinant colonies or plaques. We recommend a five-fold dilution of the ligation mix, and adding not more than 1/10 of the diluted volume to the cells. For best results, the volume added should also not exceed 10% of the volume of the competent cells that you are using.

Answer Id: E3098

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Product FAQ

How can I tell whether or not a reagent bottle is pressurizing correctly when using the Procise™ System?

Answer

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: E1261

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Product FAQ

How often should the sound of the vacuum assist be heard on the 433A?

Answer

At most, once or twice a day. If more frequent, there may be a gas leak. Nitrogen pressure is used to generate the vacuum, which assists the opening of the valves. If there is no apparent gas leak, then it is possible that a valve has failed and the solvent leakage has damaged the vacuum ballast. Both the vacuum system and the valve block need inspection.

Answer Id: E1315

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Product FAQ

How can I test for inhibitors in  my ligation reactions?

Answer

To test for the presence of ligation inhibitors, perform a ligation reaction in which some of the vector or insert DNA is included along with some marker DNA such as lambda DNA/Hind III Fragments. If ligation of the DNA marker fragments occurs alone but is not observed when other DNA is added, then a diffusible inhibitor is present in the vector or insert DNA.

To purify and remove inhibitors, extract the DNA with buffer-saturated phenol, then extract with chloroform:isoamyl alcohol, and precipitate with ammonium acetate and ethanol. Be sure that the DNA is free of phenol and that the phosphate concentration is less than 25 mM and the NaCl concentration is less than 50 mM. Also, be sure that the DNA is free of contaminating DNA that might compete for ligation to the insert or vector (e.g., linker fragments, DNA fragments from which the insert was completely purified).

If restriction endonucleases are present, causing redigestion of ligated products, your ligation will also be inhibited. After digestion of the vector and insert DNA, remove restriction endonucleases by extraction with buffer-saturated phenol, extraction with chloroform:isoamyl alcohol, and ethanol precipitation.

Answer Id: E4012

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Product FAQ

Which T4 DNA Ligase protocol do you recommend when ligating an insert containing one cohesive (sticky) end and one blunt end?

Answer

For cloning an insert with one cohesive end and one blunt end, use the conditions for blunt ends.  The sticky end may ligate quickly, but the blunt end ligation will still be inefficient. You should use the more stringent protocol to optimize the blunt end ligation. This usually means using more enzyme (5 U), a lower reaction temperature (14C) and a longer incubation time (16-24 hours). 

Answer Id: E4268

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Product FAQ

I want to make peptide amides, but your amide resin has no amino acid attached. Why not? Do I need to do anything special?

Answer

There is no amino acid attached because one is not needed. The amide linker has a free amine which is protected by an Fmoc group. Upon removal of the Fmoc group, an amide bond may be formed with the incoming activated amino acid. Nothing special needs to be done, although you must tell your synthesizer you are using an amide support and/or the first amino acid is not on the support. Standard cleavage protocols may be used.

Answer Id: E1236

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Product FAQ

Why is the conductivity so high in my peptide synthesis (monitored during deprotection)?

Answer

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: E1313

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Product FAQ

If ligation/transformation reactions did not yield any colonies, or if a high number of background colonies are observed, what control reactions should be used to determine the problem?

Answer

Several ligation controls may be necessary to identify the source of ligation problems.

Recommendations for problems with no colonies after transformation:
1. Test the transformation efficiency of the competent cells using a supercoiled vector, or the control DNA provided with Invitrogen™ competent cells. Perform a transformation reaction and plate the number of cells that is expected to generate 50-100 colonies per plate, based on the anticipated transformation efficiency of the competent cells. The expected number of colonies should be seen, indicating that the competent cells are transforming with high efficiency. The control DNA provided with Invitrogen™ competent cells is supercoiled monomer; vector DNA preparations that contain other forms will not transform as efficiently. Transformation efficiencies will be up to 10-fold lower for ligated vectors than for intact control DNA.
2. Restriction endonuclease-digested, re-ligated vector. Set up a ligation reaction using the same amount of vector DNA that is used in the experimental ligations and use it to transform competent cells. Re-ligation of vectors with cohesive ends should result in less than or equal to 50% of the number of colonies obtained with supercoiled vector DNA, indicating that the components of the ligation reaction are working; re-ligation of vectors with blunt ends should yield 10% to 20% of the number of colonies obtained with supercoiled vector DNA. This is an appropriate control only with vectors that have been digested with a single restriction endonuclease; double-digested vectors may not re-ligate because the ends are incompatible and the small DNA fragment that is released from between the two sites is sometimes lost during ethanol precipitation of the DNA.

For observation of a high number of background colonies:
1. Restriction endonuclease-digested vector. Perform a transformation with an amount of restriction enzyme-digested vector DNA equivalent to that contained in the fraction of the ligation reaction being used for the experimental transformations. Few or no colonies should be seen, indicating complete restriction endonuclease digestion of the vector. The presence of colonies indicates incomplete digestion of the vector that will cause a background of colonies containing non-recombinant vector in the experimental transformations.
2. Restriction endonuclease-digested, dephosphorylated, re-ligated vector. Set up a ligation reaction using the same amount of vector DNA that is used in the experimental ligations and use it to transform competent cells. Few or no colonies should be observed, indicating complete dephosphorylation of the vector - a dephosphorylated vector should not be re-ligated by T4 DNA ligase.
3.  No DNA transformation control. Perform a mock transformation of competent cells, to which no DNA is added. No colonies should be seen, indicating that the selection antibiotic on the agar plates is potent and that the competent cells are pure.

Answer Id: E4010

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Product FAQ

What concentration of insert and vector do you recommend for most ligation reactions?

Answer

Recommendations would vary depending on the size of the vector and insert or the nature of the insert, but for most plasmid cloning or subcloning reactions, a vector concentration of 1-10 ng/µl is recommended. Inserts should generally be 2- to 3-fold excess in molar concentration relative to the vector.

Answer Id: E4013

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Product FAQ

How can peptide synthesis amino acid cartridges leak and spill solvents during their activation and transfer to the RV?

Answer

If these cartridges are being reused, the NMP can cause them to swell, and they no longer fit or slide well in the guideway. If the guideway or the exterior of the needles have became dirty, this can also lead to misalignment. And if you forgot to remove the metal cap, the needle cannot penetrate the septum - this may cause a spill OR stop the run.

Answer Id: E1316

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Product FAQ

How many degenerate sites are allowed in a 25-mer present at a total concentration of 1 micromolar (100 pmoles/100 mL)?

Answer

Primer mixtures with 256-fold and 32-fold degeneracies have been used [see Mack DH, Sninsky JJ (1988) Proc Natl Acad Sci USA 85:6977-6981 and Lee et al. (1988) Science 239:1288-1291.] We recommend that users synthesize pools of no more than 32- to 64-fold degenerate primers, making additional pools separately to account for all possible degeneracy. A matrix should be set up so that degeneracy is no more than 2-fold at each site, with all sites in the matrix run at the same time. Inosine may also be used for the degenerate positions in the primer. Performing touchdown PCR may help increase the specificity of degenerate primer PCR amplification.

Answer Id: E1103

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Product FAQ

What causes synthesized peptides to be over or under the expected weight when post-cleavage mass spectral analysis is performed? What causes multiple peaks for one peptide, by RP-HPLC?

Answer

Overweight: Most often reflects failure to remove all the side-chain protecting groups from all amino acids ("incomplete cleavage"). If insufficient amounts of scavengers were used, these protecting groups may reattach, OR permanently attach to another amino acid. If this occurs, it may be necessary to modify the system used for cleavage. Increased time, better mixing, or increased scavengers may be needed. This may be best determined by trial and error.
Underweight: Usually reflects a problem during the synthesis. If the run was done with conductivity or UV monitoring during Fmoc removal, it may be possible to modify the subsequent synthesis to avoid problems at specific areas of the sequence. An analysis of the probable secondary structure of the peptide chain may also be helpful for future strategies.

Answer Id: E1318

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Product FAQ

Why do I sometimes get low cDNA yield when using SuperScript™ Reverse Transcriptase?

Answer

Low cDNA yield can result due to several different reasons. Please see a few listed below:

(1) Poor quality mRNA: visualize total RNA on a denaturing gel to verify that the 28S and 18S bands are sharp. OD 260:280 ratio should be 1.7.

(2) Template degraded by RNase contamination: maintain aseptic conditions.

(3) Inhibitors of SuperScript™ II RT may be present: remove inhibitors by ethanol precipitation of the RNA preparation before the first-strand reaction. Include a 70% (v/v) ethanol wash of the RNA pellet. Test for the presence of inhibitors by mixing 1 μg control RNA and comparing yields of first-strand cDNA.

(4) RNA preparation may have coprecipitated with polysaccharides: precipitate RNA with lithium chloride to purify RNA.

(5) mRNA concentrations were overestimated: quantitate the mRNA concentrations by measuring the A260 if possible.

(6) If using 32P-isotope, it may be too old: use isotope less than 2 weeks old.

(7) Not enough enzyme was used: use 200 U SuperScript™ II RT/μg RNA.

(8) SuperScript™ II RT activity was decreased by incorrect reaction temperature: perform the first-strand reaction at a temperature between 37 degrees C and 50 degrees C.

(9) DTT was not added to first-strand reaction.

(10) TCA precipitations were performed incorrectly: adequately dry GF/C filters before immersion into scintillant.

(11) SuperScript™ II RT was improperly stored: store at -20 degrees C. Do not store the enzyme at -70 degrees C.

(12) The reaction volume was too large: the reaction should be done in volumes less than or equal to 50 μL.

Answer Id: E4015

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Product FAQ

How can I determine if my ligase is still active?

Answer

Ligation reagents may be tested by performing a ligation reaction with a molecular size marker such as the 1 Kb DNA Ladder or lambda DNA/Hind III Fragments. Compare the ligation reaction products to unlighted DNA on an agarose gel. The ligation reaction should contain a high molecular weight smear and few low-molecular weight bands. If the marker ligation does not work, use fresh ligase.

Another reason for low activity could be degradation of the ATP in the reaction buffer; use 5X ligase buffer that is less than 24 months old. The buffer should be stored at -20°C.

Answer Id: E4011

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