FAQs

pGEM^®-T and pGEM^®-T Easy Vector Systems

See Technical Resources for more information.

1. What are the pGEM^®-T and pGEM^®-T Easy Vector Systems?
2. What is the difference between the pGEM^®-T and the pGEM^®-T Easy Vectors?
3. How do the pGEM^®-T and pGEM^®-T Easy Vector Systems work?
4. What types of ends do various thermostable polymerases leave?
5. How are recombinant plasmid-containing colonies screened?
6. Are there limitations to the blue/white colony screening strategy?
7. What results can I expect using the pGEM^®-T and pGEM^®-T Easy Vector Systems?
8. Why are the pGEM^®-T and pGEM^®-T Easy Vectors not available without ligase?
9. What are the optimal ligation conditions for cloning a PCR product?
10. Do I need to gel-purify the PCR product?
11. What controls can I perform if the fragment of interest is not recovered?
12. What might be the problem if the controls work as predicted but the fragment of interest is not recovered?

1. What are the pGEM^®-T and pGEM^®-T Easy Vector Systems?
   The pGEM^®-T and pGEM^®-T Easy Vector Systems facilitate the direct cloning of PCR products into a convenient plasmid vector. The systems include pGEM^®-T Vector Control DNA, T4 DNA Ligase, 2X Rapid Ligation Buffer and pGEM^®-T or pGEM^®-T Easy Vector. These vectors have been linearized and have had 3′-terminal thymidine nucleotides added.

2. What is the difference between the pGEM^®-T and the pGEM^®-T Easy Vectors?
   The only difference between the two vectors is in the multiple cloning region (MCR). The MCR of the pGEM^®-T Easy Vector contains recognition sequences for the restriction enzymes NotI and EcoRI on either side of the insert. This allows the insert DNA to be removed with a single restriction digest using either of these enzymes. The enzyme BstZ I may be used in a single restriction digest to excise the insert from both the pGEM^®-T and the pGEM^®-T Easy Vector. Both vectors are available in systems with (System II) and without (System I) competent cells.
   pGEM^®-T Vector map                                        pGEM^®-T Easy Vector map

3. How do the pGEM^®-T and pGEM^®-T Easy Vector Systems work?
   The pGEM^®-T and pGEM^®-T Easy Vector Systems take advantage of the template-independent addition of a single deoxyadenosine at the 3′-end of PCR products by some thermostable DNA polymerases. These PCR fragments are ligated to linearized vector DNA that has been cleaved at an EcoR V site and had a single 3′-terminal thymidine added to both ends. By using these vectors, PCR products can be directly cloned without further enzymatic manipulation while taking advantage of the high efficiency of a cohesive-end ligation.

4. What types of ends do various thermostable polymerases leave?
   The use of different thermostable DNA polymerases results in different types of 3′ ends on the PCR products. In general, use of proofreading enzymes, which have a 3′-> 5′ exonuclease activity, will result in blunt-ended PCR products, and use of nonproofreading enzymes will result in PCR products containing a 3′-A. Please see the table below for information on specific polymerases.

   	Taq	Tfl	Tth	Tli (Vent®)	Deep Vent®	Pfu	Pwo	"Long PCR" enzyme mixes†
   Resulting DNA ends	3′-A	3′-A	3′-A	>95% Blunt	>95% Blunt	Blunt	N.A.	Varies
   5′->3′ exonuclease activity	Yes	Yes	Yes	No	No	No	No	Yes
   3′->5′ exonuclease activity	No	No	No	Yes	Yes	Yes	Yes	Yes

   ^† Long PCR mixes generally contain a proofreading enzyme, which will cleave a 3′-A, and a nonproofreading enzyme, which will add a 3′-A. The efficiency of A addition will depend on the exact mix used.
   N.A.: not available

5. How are recombinant plasmid-containing colonies screened?
   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.

6. Are there limitations to the blue/white colony screening strategy?
   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.

7. What results can I expect using the pGEM^®-T and pGEM^®-T Easy Vector Systems?
   The cloning efficiency of a given insert cannot be known a priori. However, using the pGEM^®-T or pGEM^®-T Easy Vector, pGEM^®-T Vector Control DNA, high efficiency competent cells (>10⁸cfu/μg) and the protocol provided should produce ~100 colonies. Of these colonies, >60% should be white, and >80% of the white colonies should contain plasmid with pGEM^®-T Vector Control DNA insert.

8. Why are the pGEM^®-T and pGEM^®-T Easy Vectors not available without ligase?
   Promega T4 DNA Ligase (Cat.# M1801, M1804, M1794) has been tested for nuclease contamination. Other sources of ligase may contain exonuclease activity or other contaminants that could remove overhanging terminal nucleotides and result in higher levels of nonrecombinants.

9. What are the 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.

10. Do I need to gel-purify the PCR product?
    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.

11. What controls can I  perform if the fragment of interest is not recovered?

    • Plate untransformed competent cells.
      Growth of colonies indicates inactive ampicillin in the plates, contamination with a different plasmid that contains an ampicillin-resistance cassette or an ampicillin-resistant strain of bacteria.
    • Check transformation efficiency of competent cells by transforming with an intact plasmid and calculating colony forming units (cfu) per μg.
      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 dilution to 1000μl 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		103ng
      	X		=	106cfu/μg
      1ng of DNA plated		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.

    • 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 Ts may be missing from the vector. This may be observed if the ligase used is contaminated with a nuclease. T4 DNA Ligase (Cat.# M1801, M1804, M1794) is quality controlled to be free of contaminating nucleases; substituting T4 DNA ligase from another source is not recommended.
    • The pGEM^®-T or pGEM^®-T Easy Vector ligated to pGEM^®-T Vector Control 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.

12. What might be the problem if the controls work as predicted but the fragment of interest is not recovered?

    • Ligation at room temperature for 1 hour will be sufficient for the efficient cloning of most inserts. However, for maximum efficiency, allow the reaction to continue overnight at 4°C.
    • There may be a contaminant in the insert preparation that is removing the 3′-terminal thymidine or inhibiting ligation and/or transformation. To test this, mix the insert of interest with the pGEM^®-T Vector Control DNA before performing the control ligation. If this lowers the colony number for control ligations, the insert preparation may need to be cleaned up or remade. If this causes an unusually high number of blue colonies, the insert may contain a nuclease that removes the 3′-terminal thymidine from the pGEM^®-T or pGEM^®-T Easy Vector.
    • The insert of interest may not be suitable for ligation. This is commonly seen with gel-purified insert DNA that has been overexposed to UV light. Overexposure to UV light can form pyrimidine dimers, which can interfere with ligation. If this occurs the DNA must be repurified.
    • Thermostable polymerases with proofreading activity do not produce inserts with the single A-overhang necessary for cloning in the pGEM^®-T or pGEM^®-T Easy Vector. Single A-overhangs can be introduced to the insert of interest with the addition of Taq DNA Polymerase and nucleotides. Please see the pGEM^®-T and pGEM^®-T Easy Vector Systems Technical Manual (#TM042) for details on this procedure.
    • Highly repetitive sequences may be unstable and form deletions or rearrangements upon propagation. If the fragment of interest is deleted or rearranged with a high frequency, it may be necessary to transform an E. coli strain that is recombination-deficient such as SURE^® cells.