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Changes over time for: Division
6.3.
Timeline of Changes
This timeline shows the different versions taken from EUR-Lex before exit day and during the implementation period as well as any subsequent versions created after the implementation period as a result of changes made by UK legislation.
The dates for the EU versions are taken from the document dates on EUR-Lex and may not always coincide with when the changes came into force for the document.
For any versions created after the implementation period as a result of changes made by UK legislation the date will coincide with the earliest date on which the change (e.g an insertion, a repeal or a substitution) that was applied came into force. For further information see our guide to revised legislation on Understanding Legislation.
Status:
EU Directives are published on this site to aid cross referencing from UK legislation. Since IP completion day (31 December 2020 11.00 p.m.) no amendments have been applied to this version.
[6.3. Analysis of the PCR product U.K.
6.3.1. Resolve PCR amplicons by agarose gel electrophoresis. Run at least 12 µl of amplified DNA reaction mixture from each sample mixed with 3 µl loading buffer (Appendix 6) in 2,0 % (w/v) agarose gels in tris-acetate-EDTA (TAE) buffer (Appendix 6) at 5 to 8 V per cm. Use an appropriate DNA marker, e.g. 100 bp ladder. U.K.
6.3.2. Reveal DNA bands by staining in ethidium bromide (0,5 mg per L) for 30 to 45 min taking appropriate precautions for handling this mutagen . U.K.
6.3.3. Observe stained gel under short wave UV transillumination (e.g. λ = 302 nm) for amplified PCR products of the expected size (Appendix 6) and document. U.K.
6.3.4. For all new findings/cases verify authenticity of the PCR amplicon by performing restriction enzyme analysis on a sample of the remaining amplified DNA by incubating at the optimum temperature and time with an appropriate enzyme and buffer (see Appendix 6). Resolve the digested fragments by agarose gel electrophoresis as before and observe characteristic restriction fragment pattern under UV transillumination after ethidium bromide staining and compare with the undigested and digested positive control. U.K.
Interpretation of the PCR test result:
The PCR test is negative if the C. m. subsp. sepedonicus -specific PCR amplicon of expected size is not detected for the sample in question but is detected for all positive control samples (in case of multiplex PCR with plant specific internal control primers: a second PCR-product of expected size must be amplified with the sample in question).
The PCR test is positive if the C. m. subsp. sepedonicus -specific PCR amplicon of expected size and restriction pattern (when required) is detected, providing that it is not amplified from any of the negative control samples. Reliable confirmation of a positive result can also be obtained by repeating the test with a second set of PCR primers (section 9.3).
Note: U.K.
Inhibition of the PCR may be suspected if the expected amplicon is obtained from the positive control sample containing C. m. subsp. sepedonicus in water but negative results are obtained from positive controls with C. m. subsp. sepedonicus in potato extract. In multiplex PCR protocols with internal PCR controls, inhibition of the reaction is indicated when neither of the two amplicons are obtained.
Contamination may be suspected if the expected amplicon is obtained from one or more of the negative controls.]
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