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As a new one in the laboratory, it is not a good job to screen out positive plants from a bunch of plants with a low conversion rate. Firstly, DNA must be extracted from a large number of samples one by one, and then the foreign genes will be detected by PCR. However, the results are often blanks and bands with a few items occasionally, but it is impossible to determine whether there are missed detections or false detections. . Is it very helpless to face such experimental process and results? Don’t worry, brother teaches you how to screen out transgenic positive plants easily and accurately.

Step 1

Design detection primers

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Determine the endogenous gene and exogenous gene to be detected according to the sample to be tested, and select a representative 100-500bp sequence in the gene for primer design. Good primers can ensure the accuracy of the detection results and shorten the detection time (see the appendix for commonly used detection primers).

Notice:The newly designed primers need to optimize the reaction conditions and verify the accuracy, precision and detection limit of the detection before large-scale detection.

Step 2

Design experimental protocol

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Positive control: Use the purified DNA containing the target fragment as a template to determine whether the PCR reaction system and conditions are normal.

Negative/blank control: Use the DNA template or ddH2O that does not contain the target fragment as a template to detect whether there is a source of contamination in the PCR system.

Internal reference control: use the primer/probe combination of the endogenous gene of the sample to be tested to evaluate whether the template can be detected by PCR.

Notice:

Positive, negative/blank controls and internal control controls should be set for each test to evaluate the validity of the experimental results.

Experiment preparation

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Before use, observe whether the solution is evenly mixed. If precipitation is found, it needs to be dissolved and mixed according to the instructions before use. 2×PCR mix needs to be pipetted and mixed repeatedly with a micropipette before use to avoid uneven ion distribution.

Notice:

Take out the manual and read it carefully, and make preparations before the experiment in strict accordance with the requirements of the manual.

Step 4

Prepare PCR reaction system

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According to the experimental protocol, mix the primers, H2O, and 2×PCR mix evenly, centrifuge and distribute them to each reaction tube.

Notice:

For large-scale or long-term testing, it is recommended to use a PCR reaction system containing UNG enzyme, which can effectively avoid aerosol contamination caused by PCR products.

Step 5

Add reaction template

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Using Direct PCR technology, there is no need for tedious nucleic acid purification process, the sample template can be prepared within 10 minutes, and the corresponding PCR reaction system can be added.

Notice:

The cleavage method has better detection effect, and the obtained product can be used for multiple detection reactions.

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5.1: Direct expansion of leaves

According to the size of the picture in the manual, cut the leaf tissue with a diameter of 2-3mm and place it in the PCR reaction system.

Note: Ensure that the leaf fragments are completely immersed in the PCR reaction solution, and do not add excessive leaf tissue.

5.2: Leaf split method

Cut the leaf tissue with a diameter of 5-7mm and place it in a centrifuge tube. If you choose mature leaves, please avoid using the tissues of the main vein of the leaf. Pipette 50ul Buffer P1 lysate into a centrifuge tube to ensure that the lysate can completely immerse the leaf tissue, place it in a thermal cycler or a metal bath, and lyse at 95°C for 5-10 minutes.

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Add 50ul Buffer P2 neutralization solution and mix well. The resulting lysate can be used as a template and added to the PCR reaction system.

Note: The amount of template is between 5-10% of the PCR system, and should not exceed 20% (for example, in a 20μl PCR system, add 1-2μl of lysis solution, not more than 4μl).

Step 6

PCR reaction

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After centrifuging the PCR reaction tube, it is placed in a PCR instrument for amplification.

Notice:

The reaction uses non-purified template for amplification, so the number of amplification cycles is 5-10 more cycles than when using purified DNA template.

Step 7

Electrophoresis detection and result analysis

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M: 100bp DNA Ladder

1\4: Purified DNA method

2\5: Direct PCR method

3\6: Blank control

QC:

The test results of the various controls set in the experiment should meet the following conditions. Otherwise, the cause of the problem should be analyzed, and the test should be performed again after the problem is eliminated.

Table 1. Normal test results of various control groups

*When the plasmid is used as a positive control, the endogenous gene test result can be negative

Result judgment:

A. The test result of the endogenous gene of the sample is negative, indicating that the DNA suitable for ordinary PCR detection cannot be extracted from the sample or the extracted DNA contains PCR reaction inhibitors, and the DNA should be extracted again.

B. The test result of the endogenous gene of the sample is positive, and the test result of the exogenous gene is negative, indicating that DNA suitable for ordinary PCR detection is extracted from the sample, and it can be judged that the XXX gene is not detected in the sample.

C. The test result of the endogenous gene of the sample is positive, and the test result of the exogenous gene is positive, indicating that DNA suitable for ordinary PCR detection has been extracted from the sample, and the sample DNA contains the XXX gene. Confirmation experiments can be further carried out.

Step 8

Design detection primers

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After the experiment, use 2% sodium hypochlorite solution and 70% ethanol solution to wipe the experimental area to prevent environmental pollution。


Post time: Sep-08-2021