Q1：Why are there few or no growing clones in the end result?

A1：

1) The purity of the ligated fragments is poor. The PCR product is recommended to be purified by gel recovery (Cat. No.: GE706), and the concentration of the gel recovered product can be detected by electrophoresis or instrument before ligation reaction. It is not recommended to recover DNA by passing multiple tubes of samples through one column, which will cause more impurities such as EDTA and guanidine salts to remain and inhibit the reaction. Do not use TE to store the recovered gel product used for the ligation reaction.

2) The amount of ligated fragments is too much or too little. Please strictly follow the dosage recommended in the manual for system preparation.

3) The ligation system was prepared on ice. Low temperature will reduce the Topo enzyme ligation efficiency, and the reaction system needs to be prepared at room temperature. It is recommended to turn immediately, do not put on ice or refrigerator.

4) Improper conversion operation. Please strictly follow the transformation process in this manual or the corresponding competent instructions (pay attention to the heat shock temperature and heat shock time) to carry out the experiment.

5) Competent efficiency is low. The efficiency of self-made competent cells may decrease with the increase of storage time. It is recommended to use fresh competent cells or high-efficiency commercial competent cells as much as possible (Cat. No.: SCC01).

6) The tablet resistance is used incorrectly. The carrier resistance is Amp, and the recommended concentration is 100 ng/mL.

Q2：Why do most of the clones in the results contain no inserts?

A2：

1) PCR products contain more non-specific amplification products. It is recommended to optimize primers and PCR conditions to improve product specificity; PCR products need to be purified by cutting gel; non-specific small fragments are encapsulated in the target band and are not separated, use low-concentration agarose gel, low-voltage long-term electrophoresis separation; the number of clones to screen for positive clones.

2) Environmental pollution introduces miscellaneous fragments. It is recommended to periodically replace the electrophoresis solution, clean the gel cutting instrument, and use sterilized experimental consumables, etc. Nucleic acid cleaner (Cat. No.: ND709) can be used to clean the experimental environment.

3) Cut the glue at the wrong UV wavelength. It is recommended to cut the gel under long-wavelength (365 nm) UV light (the time should not exceed 5 min), and short-wavelength (254 nm) UV light can easily cause DNA damage.

Q3：Why do positive clones show deletions or mutations at both ends of the target fragment after the positive clones are sent for testing?

A3：

1) The enzyme products used have low fidelity. It is recommended to replace PCR enzymes with higher fidelity for amplification.

2) Poor quality of primers. Chemically synthesized primers may have a certain base deletion or mutation. It is recommended to clone or re-synthesize primers.

3) The PCR reaction was not extended sufficiently. It is recommended to extend the final extension time.

4) Cut the glue at the wrong UV wavelength. It is recommended to cut the glue under long-wavelength (365 nm) UV, and the cutting time should not exceed 5 min.

Manual: Click to download

[1] Wang N, Song G, Zhang F, et al. Characterization of the WRKY Gene Family Related to Anthocyanin Biosynthesis and the Regulation Mechanism under Drought Stress and Methyl Jasmonate Treatment in Lycoris radiat. Molecular Sciences. 2023,24(3):2423.