How can I test for inhibitors in  my ligation reactions?

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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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How can I improve peak resolution between ASP and DTT or ASN when using the Procise™ System?

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You can move ASP (and GLU) away from DTT and to later retention times by reducing slightly the pH of Solvent A3. This is best accomplished by adding a small amount of TFA (R3)--about 50 to 100 ul/liter of Solvent A3. In the Procise™ System cLC, when ASP and ASN are too close together, the problem can usually be resolved by replacing the guard column (part no. 401883). Decreasing initial %B in the gradient (e.g., from 10% to 8%) will move both DTT and ASP to later retention times.

Answer Id: E1253

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

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

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

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

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

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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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What are the recommended conditions for blunt-ended ligations?

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Answer

Generally, ligations are done in a 20 ?l volume. Use a total of 100 to 1000 ng of DNA with an insert to vector ratio of 3:1. Add 1.0 units (Weiss) to the reaction. Incubate at room temperature for 4 h or overnight at 14-16°C.

Ideally, assemble several reactions with varying ratios of vector:insert (i.e. 3:1, 5:1, 10:1, 20:1, etc.) to determine the optimal ratio for ligation.

Thermo Fisher Scientific offers T4 DNA ligase at two concentrations: 1 U/?l (15224-017) and 5 U/ul (15224-041). When performing blunt or TA cloning ligations, the higher concentration of ligase is generally preferred since ligating a blunt or single base overhang requires more enzyme.

Answer Id: E2951

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Do both my insert and vector need to be phosphorylated for ligation?

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Answer

At least one molecule in a ligase reaction (i.e., insert or vector) must be phosphorylated. Ligation reactions are dependent on the presence of a 5' phosphate on the DNA molecules. The ligation of a dephosphorylated vector with an insert generated from a restriction enzyme digest (phosphorylated) is most routinely performed. Although only one strand of the DNA ligates at a junction point, the molecule can form a stable circle, providing that the insert is large enough for hybridization to maintain the molecule in a circular form.

Answer Id: E4014

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What is the difference between T4 DNA Ligase and E. coli DNA Ligase?

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Answer

The main difference between the 2 enzymes is that E. coli DNA Ligase cannot ligate blunt dsDNA fragments. Both ligases can be used to repair single stranded nicks in duplex DNA and to perform cohesive or sticky end ligations. E. coli DNA Ligase is generally used to seal nicks during second strand cDNA synthesis, since T4 DNA Ligase could result in formation of chimeric inserts.

Answer Id: E7647

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What are some of the problems associated with sticky-end cloning?

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Answer

The amplified DNA needs to be purified from the PCR mixture components prior to cloning. The dNTPs carried over from the PCR are competitive inhibitors for ATP in the ligation reaction.

If during synthesis of the PCR primers their chemical integrity has been compromised by either a base substitution or modification, the enzyme recognition site may in actuality not exist. If this is the case, PCR products will be resistant to digestion with restriction enzymes. It may be necessary to use a higher concentration of the restriction enzyme and to incubate at the appropriate temperature overnight to ensure cutting.

Answer Id: E1102

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What can cause broad dips or cyclical peaks in the baseline when using the Procise™ System?

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Answer

This can be due to pump irregularities, often caused by worn seals. A more or less regular "sawtooth" pattern is usually indicative of a failed dynamic mixer.

Answer Id: E1251

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

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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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Which is better to use, T4 or E. coli DNA ligase?

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Answer

It depends on your application. For ligation of dsDNA fragments with cohesive ends, either enzyme can be used. E. coli DNA ligase requires the presence of beta-NAD, while T4 DNA ligase requires ATP. However, only T4 DNA ligase can join blunt-ended DNA fragments - E. coli ligase is unable to join such fragments.

E. coli DNA ligase is generally used to eliminate nicks during second-strand cDNA synthesis. T4 DNA ligase should not be substituted for E. coli DNA ligase in second-strand synthesis because of its capability for blunt end ligation of the ds cDNA fragments, which could result in formation of chimeric inserts.

Answer Id: E2949

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

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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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