BS ISO 18074:2015 pdf download – Textiles — Identification of some animal fibres by DNA analysis method — Cashmere, wool, yak and their blends.
9.6  Detection and confirmation of DNA amplification 9.6.1  Preparation 9.6.1.1  Put the 0,9 g of agarose in the Erlenmeyer flask. Then, add 60 ml of electrophoretic migration buffer solution (7.19) to the flask. Melt the agarose completely by heating. Pour the melted agarose into a gel container and set a comb in the gel. Keep the gel container with the gel at room temperature for 30 min or more and materialize the gel. 9.6.1.2  Take out the comb carefully and create wells in the gel. Set the gel container in the electrophoretic migration unit. Pour the buffer solution (7.19) into the gel container to a depth of 3 mm to 5 mm from the surface of the gel to solution surface. Take out the comb very carefully to avoid damaging the wells. 9.6.2  Electrophoretic migration test 9.6.2.1  Take 10 μl of PCR reaction solution (9.5.3.4) from the preserved micro tube using the micro pipette and put it into a new micro tube. 9.6.2.2  Add 2 μl of buffer solution (7.18) to the micro tube (9.6.2.1). Mix the contents of the micro tube pipetting. 9.6.2.3  Put the mixed solution (9.6.2.2) into the wells using a micro pipette. 9.6.2.4  Add 5 μl of gel electrophoretic migration marker (7.16) to one of the wells in the gel. 9.6.2.5  Apply a voltage of 100 V ± 50 V to the electrophoretic migration. Turn off the voltage when the dye in the electrophoresis loading buffer is migrated by 2/3 of whole migration distance.
9.6.2.6  Put the gel in a generic plastic box with a resealable lid (6.12). Then, add the ethidium bromide solution of 100 ml in it, and immerse the gel for 5 min and drain the solution. To increase the signal-to- noise ratio, immerse the gel in pure water for approximately 2 min to wash off the excess dye in the gel. 9.6.2.7  Take the gel out from the generic plastic box with a resealable lid (6.12) by a pallet and place it on the UV illuminator (6.10). Apply ultraviolet rays to the gel and observe the grow line due to the DNA, take a photograph of it in the photo booth (6.11). 10 Verification 10.1 General The preparation of specimen for the verification is given in 10.2 to 10.6. 10.2 No amplification verification after extraction process (negative verification) In the DNA extraction process described in 9.3, no animal fibre specimen is used. The DNA reaction solution for this purpose is produced by running through all the processes without a specimen. The frequency of the verification test is once per testing practice. There should be no DNA, so amplification shall not be observed for this verification test.