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pGEM-T and pGEM-T Easy Troubleshooting Guide

Previous steps:

    Result:

    This troubleshooting guide will help you resolve your issues with the pGEM®-T and pGEM®-T Easy Vector Systems. Select one of the problems below to begin.

    Did you perform the controls recommended (e.g., transformation control, positive control, background control)?

    Does the positive control reflect this same lower-than-expected number of white colonies?

    How did the positive control perform?

    Did you perform a transformation control to confirm the efficiency of the competent cells?

    Contact a member of our Global Technical Services team or e-mail: techserv@promega.com for further assistance.
    The following information is helpful to provide when calling, chatting or sending an e-mail to Technical Services:
    Did you make any changes to the protocol in the Technical Manual?
    Did the controls perform as expected?
    How was your insert purified prior to ligation?
    How was the concentration of your insert determined?
    Have you performed the PCR and cloning more than once?
    Have you seen the same results each time?
    What was the transformation efficiency of your competent cells?

    Without the use of controls, it is difficult to diagnose the issue. The issue could be low-competency cells, poor ligation or other reagent-based problems (see the information below for more details). Please perform the recommended controls, and return to the troubleshooting guide.

    Recommended controls for T-vector cloning

    • Positive control
      The pGEM®-T or pGEM®-T Easy Vector ligated to Control Insert DNA and transformed into high-efficiency competent cells (>108cfu/μg) following the protocol provided should produce ~100 colonies. Of these colonies, >60% should be white and contain plasmid with insert. Observing only 20–40 blue colonies (very few or no white colonies) from this control is a strong indication of a ligation problem.
    • Negative control (pGEM®-T or pGEM®-T Easy Vector alone in a ligation reaction)
      If, in the absence of pGEM®-T Vector Control DNA or the PCR product, more than 20–40 blue colonies are seen (following the suggested protocol and using competent cells of >108cfu/μg efficiency), then the T overhangs may be missing from the vector, a consequence of ligase being contaminated with a nuclease. The supplied T4 DNA Ligase is quality controlled to be free of contaminating nucleases; substituting T4 DNA ligase from another source is not recommended.

    Additional controls

    • Plate untransformed competent cells.
      Growth of colonies after plating untransformed cells indicates inactive ampicillin in the plates, contamination with a different plasmid that contains an ampicillin-resistance cassette or an ampicillin-resistant strain of bacteria.
    • Assess transformation efficiency of competent cells
      Transform competent cells with an intact plasmid and calculate the resulting colony forming units (cfu) per microgram. For example, a plasmid stock at 1μg/μl is diluted 1:100, and 1μl of this solution is used for a 100μl transformation. This transformation is diluted to 1,000μl with SOC, and 100μl of this dilution is plated. After overnight incubation 1,000 colonies are counted. The transformation efficiency can be calculated as:
      total number of colonies counted
      total amount of DNA plated

      Where the total amount of DNA plated is the amount of DNA in the transformation reaction divided by appropriate dilution factors. In this case, 10ng of DNA is used in the transformation. After diluting to 1,000μl with SOC, the solution contains 10ng of DNA/ml. One tenth of this volume is plated, so a total of 1ng of DNA is plated. The final transformation efficiency is:
      1,000 colonies 

      1ng of DNA plated
      ×
      103ng

      µg
      = 106cfu/μg
      Note: Only cells producing >108cfu/μg should be used for pGEM®-T and pGEM®-T Easy Vector System transformations. If the transformation results in few or no colonies, the efficiency of the competent cells is too low or the transformation procedure was improperly followed.

     For more information, see the pGEM®-T and pGEM®-T Easy Technical Manual #TM042.

    Did all the controls perform as expected (i.e., few to no colonies with the background control, approximately 100 colonies with the positive control and a transformation efficiency of at least 108cfu/µg with the transformation control)?

    Repeat experiment using the supplied Control Insert DNA. See below for additional information on controls.

    Recommended controls for T-vector cloning

    • Positive control
      The pGEM®-T or pGEM®-T Easy Vector ligated to Control Insert DNA and transformed into high-efficiency competent cells (>108cfu/μg) following the protocol provided should produce ~100 colonies. Of these colonies, >60% should be white and contain plasmid with insert. Observing only 20–40 blue colonies (very few or no white colonies) from this control is a strong indication of a ligation problem.
    • Negative control (pGEM®-T or pGEM®-T Easy Vector alone in a ligation reaction)
      If, in the absence of pGEM®-T Vector Control DNA or the PCR product, more than 20–40 blue colonies are seen (following the suggested protocol and using competent cells of >108cfu/μg efficiency), then the T overhangs may be missing from the vector, a consequence of ligase being contaminated with a nuclease. The supplied T4 DNA Ligase is quality controlled to be free of contaminating nucleases; substituting T4 DNA ligase from another source is not recommended.

    Additional controls

    • Plate untransformed competent cells.
      Growth of colonies after plating untransformed cells indicates inactive ampicillin in the plates, contamination with a different plasmid that contains an ampicillin-resistance cassette or an ampicillin-resistant strain of bacteria.
    • Assess transformation efficiency of compentent cells
      Transform compentent cells with an intact plasmid and calculate the resulting colony forming units (cfu) per microgram. For example, a plasmid stock at 1μg/μl is diluted 1:100, and 1μl of this solution is used for a 100μl transformation. This transformation is diluted to 1,000μl with SOC, and 100μl of this dilution is plated. After overnight incubation 1,000 colonies are counted. The transformation efficiency can be calculated as:
      total number of colonies counted
      total amount of DNA plated

      Where the total amount of DNA plated is the amount of DNA in the transformation reaction divided by appropriate dilution factors. In this case, 10ng of DNA is used in the transformation. After diluting to 1,000μl with SOC, the solution contains 10ng of DNA/ml. One tenth of this volume is plated, so a total of 1ng of DNA is plated. The final transformation efficiency is:
      1,000 colonies 

      1ng of DNA plated
      ×
      103ng

      µg
      = 106cfu/μg
      Note: Only cells producing >108cfu/μg should be used for pGEM®-T and pGEM®-T Easy Vector System transformations. If the transformation results in few or no colonies, the efficiency of the competent cells is too low or the transformation procedure was improperly followed.

     For more information, see the pGEM®-T and pGEM®-T Easy Technical Manual #TM042.

    There are several possibilities that may explain the less-than-optimal performance of the positive control. They include:
    • Improper dilution of the 2X Rapid Ligation Buffer. Use 5 μl of buffer in a 10μl ligation reaction. Make sure to mix the 2X Rapid Ligation Buffer well prior to dispensing into the ligation reaction.
    • Improper ligation conditions. Incubate the ligation reaction at the correct temperature. Temperatures >28 °C give rise to increased background
    and fewer recombinants.
    • Nuclease contamination. The T overhangs may have been removed, thus preventing the ligation of A-tailed PCR products. Avoid introducing nucleases that may degrade T overhangs to the reagent tubes when using the pGEM®-T or pGEM®-T Easy Vector Systems. Use only the T4 DNA Ligase provided with the system, which has been tested for minimal exonuclease activity.
    • Failed ligation reaction. This may be a consequence of the reaction buffer or ligase. The 2X Rapid Ligation Buffer contains ATP, which degrades during temperature fluctuations. Avoid multiple freeze-thaw cycles by making single-use aliquots of the buffer. Use a fresh vial of buffer. To test the activity of the ligase and buffer, set up a ligation with ~20ng of DNA markers (e.g., Lambda DNA/HindIII Markers, Cat.# G1711). Compare ligated and unligated DNA on a gel, and check that the fragments have been religated into high-molecular-weight material.

    Do your experimental ligations yield blue colonies that vary in color from light blue to dark blue?

    Repeat experiment using the supplied Control Insert DNA. See below for additional information on controls to use.

    Recommended controls for T-vector cloning

    • Positive control
      The pGEM®-T or pGEM®-T Easy Vector ligated to Control Insert DNA and transformed into high-efficiency competent cells (>108cfu/μg) following the protocol provided should produce ~100 colonies. Of these colonies, >60% should be white and contain plasmid with insert. Observing only 20–40 blue colonies (very few or no white colonies) from this control is a strong indication of a ligation problem.
    • Negative control (pGEM®-T or pGEM®-T Easy Vector alone in a ligation reaction)
      If, in the absence of pGEM®-T Vector Control DNA or the PCR product, more than 20–40 blue colonies are seen (following the suggested protocol and using competent cells of >108cfu/μg efficiency), then the T overhangs may be missing from the vector, a consequence of ligase being contaminated with a nuclease. The supplied T4 DNA Ligase is quality controlled to be free of contaminating nucleases; substituting T4 DNA ligase from another source is not recommended.

    Additional controls

    • Plate untransformed competent cells.
      Growth of colonies after plating untransformed cells indicates inactive ampicillin in the plates, contamination with a different plasmid that contains an ampicillin-resistance cassette or an ampicillin-resistant strain of bacteria.
    • Assess transformation efficiency of compentent cells
      Transform compentent cells with an intact plasmid and calculate the resulting colony forming units (cfu) per microgram. For example, a plasmid stock at 1μg/μl is diluted 1:100, and 1μl of this solution is used for a 100μl transformation. This transformation is diluted to 1,000μl with SOC, and 100μl of this dilution is plated. After overnight incubation 1,000 colonies are counted. The transformation efficiency can be calculated as:
      total number of colonies counted
      total amount of DNA plated

      Where the total amount of DNA plated is the amount of DNA in the transformation reaction divided by appropriate dilution factors. In this case, 10ng of DNA is used in the transformation. After diluting to 1,000μl with SOC, the solution contains 10ng of DNA/ml. One tenth of this volume is plated, so a total of 1ng of DNA is plated. The final transformation efficiency is:
      1,000 colonies 

      1ng of DNA plated
      ×
      103ng

      µg
      = 106cfu/μg
      Note: Only cells producing >108cfu/μg should be used for pGEM®-T and pGEM®-T Easy Vector System transformations. If the transformation results in few or no colonies, the efficiency of the competent cells is too low or the transformation procedure was improperly followed.

     For more information, see the pGEM®-T and pGEM®-T Easy Technical Manual #TM042.

    Recovering a control insert of the correct size indicates the pGEM®-T or pGEM®-T Easy Vector System reagents are performing as they should. However, there are many reasons why the correct experimental insert was not recovered. Find more information below for possible explanations.

    Optimal ligation conditions for cloning a PCR product
    The optimal insert:vector ratio for a particular construct must be determined empirically. Although a 1:1 (insert:vector) molar ratio is often optimal, molar ratios ranging from 1:8 to 8:1 have been successfully used. A range of ratios should be tested for each experiment. Typical ligation conditions involve 5μl of 2X Rapid Ligation Buffer, 50ng of plasmid DNA, 1 Weiss unit of T4 DNA Ligase and insert DNA in a 10μl total volume. The ligation can be incubated at room temperature for one hour or at 4°C overnight. At either temperature, any vector lacking T overhangs will recircularize and produce blue colonies. Ligation at room temperature for 1 hour will be sufficient for the efficient cloning of most inserts. However, for maximal efficiency, allow the ligation reaction to continue overnight at 4°C.

    Gel purify the PCR product before cloning
    If a single amplification product is generated, as determined by agarose gel analysis, gel purification may not be necessary. However, even if no extraneous bands are visible, there may be some primer dimers present in the reaction. Even a very small mass of primer dimer corresponds to a large molar quantity. This can result in unacceptably high numbers of clones containing primer dimer instead of the fragment of interest. For this reason, gel purification should be considered.

    Cloning the products of proofreading enzymes such as Tli DNA polymerase
    Successful ligation into T vectors requires that the insert possess single deoxyadenosine overhangs on the 3′ ends. Enzymes that possess 3′→5′ exonuclease activity ("proofreading" activity) produce blunt-ended fragments. Single deoxyadenosine overhangs can be added following amplification to produce the required overhangs on the products of proofreading enzymes. Additional dATP and Taq DNA polymerase can be added to the reactions during the final cycles or in a subsequent reaction.

    The transformation control gives information on the efficiency of the competent cells used.
    If the efficiency is less than 1 × 108 cfu/μg, the number of colonies will be low. Perform a transformation control, and if the transformation efficiency is <1 × 108 cfu/μg, prepare or purchase new competent cells. See below for more information on the control ligations used with the pGEM®-T or pGEM®-T Easy Vector Systems.

    Recommended controls for T-vector cloning

    • Positive control
      The pGEM®-T or pGEM®-T Easy Vector ligated to Control Insert DNA and transformed into high-efficiency competent cells (>108cfu/μg) following the protocol provided should produce ~100 colonies. Of these colonies, >60% should be white and contain plasmid with insert. Observing only 20–40 blue colonies (very few or no white colonies) from this control is a strong indication of a ligation problem.
    • Negative control (pGEM®-T or pGEM®-T Easy Vector alone in a ligation reaction)
      If, in the absence of pGEM®-T Vector Control DNA or the PCR product, more than 20–40 blue colonies are seen (following the suggested protocol and using competent cells of >108cfu/μg efficiency), then the T overhangs may be missing from the vector, a consequence of ligase being contaminated with a nuclease. The supplied T4 DNA Ligase is quality controlled to be free of contaminating nucleases; substituting T4 DNA ligase from another source is not recommended.

    Additional controls

    • Plate untransformed competent cells.
      Growth of colonies after plating untransformed cells indicates inactive ampicillin in the plates, contamination with a different plasmid that contains an ampicillin-resistance cassette or an ampicillin-resistant strain of bacteria.
    • Assess transformation efficiency of compentent cells
      Transform compentent cells with an intact plasmid and calculate the resulting colony forming units (cfu) per microgram. For example, a plasmid stock at 1μg/μl is diluted 1:100, and 1μl of this solution is used for a 100μl transformation. This transformation is diluted to 1,000μl with SOC, and 100μl of this dilution is plated. After overnight incubation 1,000 colonies are counted. The transformation efficiency can be calculated as:
      total number of colonies counted
      total amount of DNA plated

      Where the total amount of DNA plated is the amount of DNA in the transformation reaction divided by appropriate dilution factors. In this case, 10ng of DNA is used in the transformation. After diluting to 1,000μl with SOC, the solution contains 10ng of DNA/ml. One tenth of this volume is plated, so a total of 1ng of DNA is plated. The final transformation efficiency is:
      1,000 colonies 

      1ng of DNA plated
      ×
      103ng

      µg
      = 106cfu/μg
      Note: Only cells producing >108cfu/μg should be used for pGEM®-T and pGEM®-T Easy Vector System transformations. If the transformation results in few or no colonies, the efficiency of the competent cells is too low or the transformation procedure was improperly followed.

     For more information, see the pGEM®-T and pGEM®-T Easy Technical Manual #TM042.

    What was the calculated colony-forming units (cfu) per microgram of DNA?

    Which of the controls gave unexpected results?

    If the positive control reaction resulted in >100 colonies, but the experimental ligation and transformation was unsuccessful, there is likely an issue with the insert.

    • If the number of colonies observed with your insert is equivalent to that with the background control, the insert might not be suitable for ligation. Check that a nonproofreading thermostable DNA polymerase was used during PCR. If a proofreading DNA polymerase was used, incubate the insert with a thermostable nonproofreading DNA polymerase like Taq, dATP and reaction buffer to add A tails to the blunt-end insert.
    • If the insert was gel-purified and UV exposure was not minimized, the pyrimidines can form dimers and inhibit ligation, resulting in fewer colonies. Repeat the PCR and purification steps, being mindful of the UV exposure.
    • A contaminant in the insert might be inhibiting transformation. To test this possibility, add a portion of the insert into one of two side-by-side positive control ligations and transform both of them. If there is an inhibitor, the positive control ligation with insert should have fewer or no colonies compared to the unadulterated positive control.
    • The insert may encode a toxic gene.

    Recommended solutions:

    • Repurify the insert using a DNA purification system like the Wizard® SV Gel and PCR Clean-Up System (Cat.# A9281).
    • Minimize the volume of insert used in the ligation. For example, using 1µl of insert rather than 3µl will decrease the concentration of inhibitor.
    • Ethanol precipitate the insert and resuspend in nuclease-free water before performing another ligation.
    • Incubate the plates with transformed cells at a lower temperature (e.g., 30°C) to slow colony growth for potentially toxic inserts. Consider cloning only a portion of the gene. Alternatively, select a lower-copy-number plasmid to minimize the toxicity of the encoded protein.

    If there are still issues with the pGEM® T or pGEM® T Easy Vector System, revisit this troubleshooting guide or contact Promega Technical Services.

    There are many reasons for the low number of white colonies. For example, the 2X Rapid Ligation Buffer may have been diluted incorrectly (less than 5μl used in a 10μl reaction), or the insert-to-vector ratio was not optimal. Alternatively, the insert might be contaminated with nucleases, which can remove the A overhang from the insert and the T overhang from the pGEM®-T and pGEM®-T Easy Vectors. To prevent this contamination, avoid introducing nucleases. Clean up or repurify the insert DNA.

    For more information on how to successfully ligate your insert using the pGEM®-T or pGEM®-T Easy Vector System, see additional information below.

    Screening recombinant plasmid-containing colonies
    Potential recombinants can be chosen by an initial blue/white colony screen. Clones may be analyzed further by techniques such as restriction enzyme analysis or small-scale PCR screening. Blue/white colony screening relies on disruption of the lacZ gene. Although the pGEM®-T Vector Control DNA will produce recombinants that generate white colonies, insertion of other DNA fragments into the lacZ coding sequence may not result in white colonies unless the fragments disrupt the lacZ reading frame. Although this tends to occur most frequently with PCR products of 500bp or less, inserts of up to 2kb have been reported to result in blue colonies. Moreover, some insert DNAs can also result in pale blue colonies. For this reason, we recommend performing a control ligation without insert DNA. For instance, the control ligation without pGEM®T Vector Control DNA may produce 20–40 blue colonies, while the experimental ligation produces 100 blue colonies. This result strongly suggests that the insert of interest has been cloned but has not sufficiently disrupted the lacZ coding sequence.

    Optimal ligation conditions for cloning a PCR product
    The optimal insert:vector ratio for a particular construct must be determined empirically. Although a 1:1 (insert:vector) molar ratio is often optimal, molar ratios ranging from 1:8 to 8:1 have been successfully used. A range of ratios should be tested for each experiment. Typical ligation conditions involve 5μl of 2X Rapid Ligation Buffer, 50ng of plasmid DNA, 1 Weiss unit of T4 DNA Ligase and insert DNA in a 10μl total volume. The ligation can be incubated at room temperature for one hour or at 4°C overnight. At either temperature, any vector lacking T overhangs will recircularize and produce blue colonies. Ligation at room temperature for 1 hour will be sufficient for the efficient cloning of most inserts. However, for maximal efficiency, allow the ligation reaction to continue overnight at 4°C.

    Gel purify the PCR product before cloning
    If a single amplification product is generated, as determined by agarose gel analysis, gel purification may not be necessary. However, even if no extraneous bands are visible, there may be some primer dimers present in the reaction. Even a very small mass of primer dimer corresponds to a large molar quantity. This can result in unacceptably high numbers of clones containing primer dimer instead of the fragment of interest. For this reason, gel purification should be considered.

    Cloning the products of proofreading enzymes such as Tli DNA polymerase
    Successful ligation into T vectors requires that the insert possess single deoxyadenosine overhangs on the 3′ ends. Enzymes that possess 3′→5′ exonuclease activity ("proofreading" activity) produce blunt-ended fragments. Single deoxyadenosine overhangs can be added following amplification to produce the required overhangs on the products of proofreading enzymes. Additional dATP and Taq DNA polymerase can be added to the reactions during the final cycles or in a subsequent reaction.

    For more information, see the pGEM®-T and pGEM®-T Easy Technical Manual #TM042.

    In some situations, the insert of interest may only partially deactivate the lacZ gene. While there are fewer white colonies than expected, the light blue colonies as well as the white ones are likely to contain your insert. Choose 10–20 colonies (both white and blue) to screen for the presence of your insert.

    If the calculated value is ≥1 × 108 cfu/μg, there may be something inhibiting the transformation or ligation of the insert. If not tested previously, perform the positive control along with the transformation control to ensure the pGEM®-T or pGEM®-T Easy Vector System reagents have not lost activity. The positive-control ligation should yield at least 100 colonies, at least 60% of which should be white. Poor positive control results often indicate a failed ligation due to several possibilities:
    • Improper ligation conditions. Incorrect dilution of the 2X Rapid Ligation Buffer. Use 5 μl of buffer in a 10 μl ligation reaction. Make sure to mix the 2X Rapid Ligation Buffer well prior to dispensing into the ligation reaction.
    • Incubate the ligation reaction at the correct temperature. Temperatures >28 °C give rise to increased background and fewer recombinants.
    • Nuclease contamination. The T overhangs may have been removed, thus preventing the ligation of A-tailed PCR products. Avoid introducing nucleases that may degrade T overhangs to the reagent tubes when using the pGEM®-T or pGEM®-T Easy Vector Systems. Use only the T4 DNA Ligase provided with the system, which has been tested for minimal exonuclease activity.
    • Reagent failure. The 2X Rapid Ligation Buffer contains ATP, which degrades during temperature fluctuations. Avoid multiple freeze-thaw cycles by making single-use aliquots of the buffer. Use a fresh vial of buffer. To test the activity of the ligase and buffer, set up a ligation with ~20 ng of DNA markers (e.g., Lambda DNA/HindIII Markers, Cat.# G1711). Compare ligated and unligated DNA on a gel, and check that the fragments have been religated into high-molecular-weight material.
    If the positive control reaction resulted in >100 colonies, at least 60% of which were white, there might be an issue with the experimental insert.
    • If the number of colonies observed with your insert is equivalent to that with the background control, the insert might not be suitable for ligation. Check that a nonproofreading thermostable DNA polymerase was used during PCR. If a proofreading DNA polymerase was used, incubate the insert with a thermostable nonproofreading DNA polymerase like Taq, dATP and reaction buffer to add A tails to the blunt-end insert.
    • If the insert was gel-purified and UV exposure was not minimized, the pyrimidines can form dimers and inhibit ligation, resulting in fewer colonies. Repeat the PCR and purification steps, being mindful of the UV exposure.
    • A contaminant in the insert might be inhibiting transformation. To test this possibility, add a portion of the insert into one of two side-by-side positive control ligations and transform both of them. If there is an inhibitor, the positive control ligation with insert should have fewer or no colonies compared to the unadulterated positive control.
    • The insert may encode a toxic gene.
    Recommended solutions:
    • Repurify the insert using a DNA purification system like the Wizard® SV Gel and PCR Clean-Up System (Cat.# A9281).
    • Minimize the volume of insert used in the ligation. For example, using 1μl of insert rather than 3μl will decrease the concentration of inhibitor.
    • Ethanol precipitate the insert and resuspend in nuclease-free water before performing another ligation.
    • Incubate the plates with transformed cells at a lower temperature (e.g., 30°C) to slow colony growth for potentially toxic inserts. Consider cloning only a portion of the gene. Alternatively, select a lower-copy-number plasmid to minimize the toxicity of the encoded protein.
    If there are still issues with the pGEM® T or pGEM® T Easy Vector System, revisit this troubleshooting guide or contact Promega Technical Services.

    At <1 × 107 cfu/μg, fewer than 10 colonies would appear on the positive control plate. Purchase or make new competent cells, and transform cells with a suitable plasmid. Transformation controls must be performed with circular plasmid DNA. Do not use the pGEM®-T or pGEM®-T Easy Vector, which is linearized, to perform the transformation control. Be sure that your competent cells have a transformation efficiency >1 × 108 cfu/μg.

    If the number of colonies was reduced for both the positive control and transformation control, it is likely the transformation efficiency of the competent cells is too low. Use fresh competent cells with an efficiency of 108cfu/µg or greater. Using cells that have been through at least one freeze/thaw cycle will reduce the transformation efficiency.

    There are several possibilities that may explain the less-than-optimal performance of the positive control. They include:

    • Improper dilution of the 2X Rapid Ligation Buffer. Use 5µl of buffer in a 10µl ligation reaction. Make sure to mix the 2X Rapid Ligation Buffer well prior to dispensing into the ligation reaction.
    • Improper ligation conditions. Incubate the ligation reaction at the correct temperature. Temperatures >28°C give rise to increased background and fewer recombinants.
    • Nuclease contamination. The T overhangs may have been removed, thus preventing the ligation of A-tailed PCR products. Avoid introducing nucleases that may degrade T overhangs to the reagent tubes when using the pGEM®-T or pGEM®-T Easy Vector Systems. Use only the T4 DNA Ligase provided with the system, which has been tested for minimal exonuclease activity.
    • Failed ligation reaction. This may be a consequence of the reaction buffer or ligase. The 2X Rapid Ligation Buffer contains ATP, which degrades during temperature fluctuations. Avoid multiple freeze-thaw cycles by making single-use aliquots of the buffer. Use a fresh vial of buffer. To test the activity of the ligase and buffer, set up a ligation with ~20ng of DNA markers (e.g., Lambda DNA/HindIII Markers, Cat.# G1711). Compare ligated and unligated DNA on a gel, and check that the fragments have been religated into high-molecular-weight material.

    The transformation control must be performed with an uncut plasmid. Using the pGEM®-T or pGEM®-T Easy Vector, which is linearized, for transformation will result in few to no colonies.

    If an uncut plasmid was used, ensure it has no contaminating salts. If this may be the issue, use another uncut vector for transformation or repurify the plasmid before using in the transformation control.

    Check the transformation efficiency calculations, and confirm that the dilution of the transformation reaction was factored into the final number. For example, if 100µl of competent cells were transformed with 0.1ng of uncut plasmid and diluted in 900µl of SOC medium, the concentration is 0.1ng DNA per 1ml of medium. If 100µl of the 1:10 dilution was diluted into another 900µl of SOC medium, the concentration of the plasmid is now 0.01ng/ml DNA (1:100 dilution of the original concentration). When 100µl of the 1:100 dilution was removed and plated, a total of 0.001ng of transformed plasmid was dispensed on the single plate (1/10 volume of the 0.01ng DNA/ml equals 0.001ng). Divide the number of colonies on the plate by the amount of plasmid DNA plated, and convert to colony-forming units per microgram of DNA.