1. Why use the GetClone™ Vector for PCR cloning instead of a T-vector?
For conducting PCR-cloning, a high fidelity polymerase is required to keep accuracy of PCR amplification. In general, high fidelity enzymes vetting correctness such as SMO-HiFi, Pfu, KOD, and Phusion are associated with proofreading activity which results in blunt ends of PCR products. GetClone™ Vector can directly accept blunt-ended PCR fragments for ligation, without the need of extra step of DNA phosphorylation. As contrast, T-vector used for TA-cloning requires DNA fragment with an extra Adenosine nucleotide added at the 3’ end which is usually generated by non-proofreading and thus less fidelity DNA polymerase like Taq polymerase.
2. Is blunt end ligation more difficult than sticky end ligation?
It depends on the length of sticky end. Generally, sticky end is easier to perform in comparison to blunt end ligation. However, a sticky end with only one extruding base as seen in the TA-cloning is probably more difficult for ligation as compared with a blunt end (also referred to NEB for NciI or EcoNI instructions).
3. After using GetClone™ Vector for PCR cloning, how can the colonies carrying the cloned fragment be selected?
In most cases of using GetClone™ Vector, colonies grown on a plate with appropriate antibiotics should be the positive clones because a bacterium accepting an empty GetClone™ Vector without an insert would fail to proliferate. To further identify the clones, it is recommended to use Colony-PCR method. In brief, the colony grown on the plate is directly picked up with a toothpick and then mixed into a PCR premix (ex.: SMOBIO TP1200
) and PCR amplification is conducted in the thermal cycler with primers (pGET-For and pGET-Rev) which are also provided with GetClone™ Vector. The expected length of PCR product should be the one of the insert plus 152 bp.
4. How do I perform sequencing when using the GetClone™ Vector?
Please use the GetClone™ Vector primers for sequence service.
5. In use of GetClone™ Vector for cloning, why are some colonies in absence of inserts?
Several reasons can be stated as follows:
Reason 1: Check your insert. If it is not blunt-end, it cannot be ligated with GetClone™ vector, as this may result in false positive results for majority of the colonies.
Reason 2: When DNA concentration of the insert is too low, the successful ligates are largely reduced in proportion, leading to an overwhelming vector background. The solution is to increase the concentration of the insert DNA.
Reason 3: If the target DNA fragments for cloning are large, such as 10kb or more, transformation efficiency depreciates after ligation. This results in fewer colonies having insertions. Therefore, it is suggested to increase the insert DNA concentration and reduce the concentration of vector DNA to re-execute ligation. It will also enhance chances to get the right clone by using higher efficiency competent cells.
Reason 4: Non-specific PCR products or/and primer dimers which might occurred during PCR amplification are supposed to compete or interfere the ligation of target PCR fragment with the vector, and thus reducing the number of correct clones. Before conducting ligation, it is therefore recommended to isolate the desired DNA fragment from other non-specific DNA by gel extraction.
6. If I did not add any insert DNA fragment for ligation with GetClone™ Vector, why are colonies being grown on the selective plate?
This is probably due to generation of mutation on the vector DNA during cloning. A mutated lethal gene can lead to false positive colonies, despite that the occurrence is rare.
7. Do you have any experiment data for cloning 12 kb fragment with GetClone™ Vector?
Yes, we do have the data as shown below. Eight colonies were picked up for plasmid isolation and PCR analysis. It was observed that seven out of eight colonies (87.5%) were positive clones which could be verified by successful amplification of 12.1 kb fragments.
8. How to count the 90% cloning efficiency of GetClone™?
The GFP report gene was PCR amplified as the insert to the GetClone™ Vector. After ligation and transformation, positive clones (with insert) presenting green fluorescence on the plate were calculated in number, and ~ 90% of total colonies did shine as observed in B-Box, demonstrating the high cloning efficiency of GetClone™ Vector .