NOVABEADS FOOD 1 DNA KIT

Transcription

1 NOVABEADS FOOD 1 DNA KIT NOVABEADS FOOD DNA KIT is the new generation tool in molecular biology techniques and allows DNA isolations from highly processed food products. The method is based on the use of fully synthetic, composite magnetic bed selectively binding nucleic acids. Specially formulated binding, washing and elution buffers allow you to get very high quality and purity DNA. An isolated DNA not fragmented during the purification process. Lack of inhibitors of subsequent reactions allows the direct use of purified nucleic acids in other molecular biology techniques, such as: PCR reaction DNA sequencing (automatic and manual) dephosphorylation phosphorylation ligation investigation of DNA-proteins interaction restriction enzymes digestion. Unlike standard beds used for isolation and purification of DNA, magnetic particles enable full automation of this process. Three modifications of the standard protocol for isolation of DNA from highly processed plant origin food are presented below. Protocols have been modified for the following products: different types of flour corn gluten ketchup.

2 Protocol for DNA isolation from flour NOVABEADS FOOD 1 DNA KIT The kit includes: Magnetic Particles Lysis Buffer Precipitation Buffer LP Buffer Binding Buffer PreWash Buffer Wash Buffer TE Buffer RNase A [10 mg/ml] Additional materials required: ethanol 80% magnetic stand vortex Attention: Mix thoroughly all buffers and vortex magnetic particles before you start work. Leave the test tube with the magnetic particles at room temperature. Before adding once again vortex thoroughly the contents of the tube. Description of the method: 1. In the 2 ml tube weight mg of flour* and add 800 µl of Lysis Buffer and 10 µl of Rnase A. Thoroughly vortex contents of the tube for few seconds and then incubate at 65 C for 20 min. 2. After incubation centrifuge the sample at room temperature for 10 min at rpm. After centrifugation, the supernatant transfer carefully to a new tube and discard the pellet. Fill the test tube to a maximum volume using Precipitation Buffer. Vortex and incubate at -20 C for 5 min. 3. Centrifuge sample at room temperature for 10 min at rpm. Supernatant removed by vigorously inverting the tube. 4. Add 250 µl of Binding Buffer to the pellet and thoroughly dissolve the pellet by vortexing. Add 20 µl of Magnetic Particles. Thoroughly vortex contents of the tube. Incubate at room temperature for 15 min. 5. Put the tube in a magnetic stand, and after collecting the magnetic particles discard the supernatant.

3 Caution! In the subsequent steps, after adding each of wash buffer (PreWash Buffer, Wash Buffer, 70% isopropanol) contents of the tube should be gently mixed by inversion until complete separation of magnetic particles from the walls of the tube. Do not vortex test tubes! 6. Magnetic particles suspend in 1 ml of PreWash Buffer. After complete separation of particles from the walls of the tube, incubate for 3 min at room temperature. Put the tube in a magnetic stand, and after collecting the magnetic particles discard supernatant. 7. Magnetic particles suspend in 1 ml of Wash Buffer. After complete separation of particles from the walls of the tube, incubate for 1 min at room temperature. Put the tube in a magnetic stand, and after collecting the magnetic particles discard supernatant. 8. Magnetic particles suspend in 1 ml of freshly prepared 80% ethanol. After complete separation of particles from the walls of the tube, incubate for 1 min at room temperature. Put the tube in a magnetic stand, and after collecting the magnetic particles discard supernatant. 9. In order to remove residual ethanol, open the tube and turn the magnetic stand upside down. Leave in this position on laboratory paper for approximately 5 min. at room temperature. 10. Magnetic particles suspend by vortexing in 50 l of TE Buffer. Incubate the mixture for 5 min at 65 C with shaking 800 rpm, and then with opened tubes for another 5 min in this same conditions. 11. After incubation, put the tube in the magnetic stand, and after collecting the magnetic particles, transfer the supernatant containing DNA into a new tube. 12. The obtained DNA can be used directly for PCR. (*) Amount of flour depends on the degree of processing.

4 Protocol for DNA isolation from corn gluten The kit includes: NOVABEADS FOOD 1 DNA KIT Magnetic Particles Lysis Buffer Precipitation Buffer Binding Buffer PreWash Buffer Wash Buffer TE Buffer RNase A [10 mg/ml] Additional materials required: Ethanol 80% Magnetic stand vortex Attention: Mix thoroughly all buffers and vortex magnetic particles before you start work. Leave the test tube with the magnetic particles at room temperature. Before adding once again vortex thoroughly the contents of the tube. Description of the method: 1. In the 1.5 ml tube weight 100 mg of corn gluten. Fill the test tube to a maximum volume using Lysis Buffer. Vortex the tube and incubate at 65 C for 10 min. 2. After incubation centrifuge the sample at room temperature for 10 min at rpm. After centrifugation, transfer the supernatant carefully to a new tube and discard the pellet. Fill the test tube to a maximum volume using Precipitation Buffer. Vortex and incubate at -20 C for 5 min. 3. Centrifuge sample at room temperature for 10 min at rpm. Supernatant remove by vigorously inverting the tube. 4. Add 250 µl of Binding Buffer to the pellet and thoroughly dissolve the pellet by vortexing. At this stage, few fractions from samples can be combined. This step allows to increase DNA concentration in the test material. Combined fractions from the two samples, will be correspond to 200 mg of corn gluten.

5 5. Regardless to the number of combined samples add 10 µl of Magnetic Particles. Vortex thoroughly the contents of the tube. Incubate at room temperature for 10 min. 6. Put the tube in a magnetic stand, and after collecting the magnetic particles discard the supernatant. Caution! In the subsequent steps, after adding each of wash buffer (PreWash Buffer, Wash Buffer, 70% isopropanol) contents of the tube should be gently mixed by inversion until complete separation of magnetic particles from the walls of the tube. Do not vortex test tubes! 7. Magnetic particles suspend in 1 ml of PreWash Buffer. After complete separation of particles from the walls of the tube, incubate for 3 min at room temperature. Put the tube in a magnetic stand, and after collecting the magnetic particles discard supernatant. 8. Magnetic particles suspend in 1 ml of Wash Buffer. After complete separation of particles from the walls of the tube, incubate for 1 min at room temperature. Put the tube in a magnetic stand, and after collecting the magnetic particles discard supernatant. 9. Magnetic particles suspend in 1 ml of freshly prepared 80% ethanol. After complete separation of particles from the walls of the tube, incubate for 1 min at room temperature. Put the tube in a magnetic stand, and after collecting the magnetic particles discard supernatant. 10. In order to remove residual ethanol, open the tube and turn the magnetic stand upside down. Leave in this position on laboratory paper for approximately 5 min at room temperature. 11. Magnetic particles suspend by vortexing in 50 l of TE Buffer. Incubate the mixture for 5 min at 65 C with shaking 800 rpm, and then with opened tubes for another 5 min in the same conditions. 12. After incubation, put the tube in the magnetic stand, and after collecting the magnetic particles, transfer the supernatant containing DNA into a new tube. 13. The obtained DNA can be used directly for PCR.

6 Protocol of DNA isolation from ketchup NOVABEADS FOOD 1 DNA KIT The kit includes: Magnetic Particles Neutralization Buffer Lysis Buffer Precipitation Buffer LP Buffer Binding Buffer PreWash Buffer Wash Buffer TE Buffer RNase A [10 mg/ml] Additional materials required: isopropanol 70% isopropanol 100% (room temperature) ethanol 70% (frozen) magnetic stand mortar mikrocentrifuge vortex Attention: Mix thoroughly all buffers and vortex magnetic particles before you start work. Leave the test tube with the magnetic particles at room temperature. Before adding once again vortex thoroughly the contents of the tube. When washing ketchup in a Neutralization Buffer and grinding in a mortar, there should be visible a clearly orange and light purple dye on the walls of the mortar. After each washing centrifuge the mixture in the same tubes. Since DNA binding to the magnetic particles (from step 11), after successive washings, the magnetic particles in PreWash Buffer, Wash Buffer and 70% isopropanol, do not vortex tubes!

7 Description of the method: STEP I Densification 1. To the mortar add 4 ml of the ketchup and 4 ml of Neutralization Buffer. The whole rub in a mortar for about 5 minutes. Transfer the mixture to four Eppendorf tubes (1 ml to one tube). Centrifuge 5 min at room temperature ( rpm). Discard the supernatant. 2. The washing procedure repeats four times. For this purpose, to each tube containing the pellet, add 1 ml of Neutralization Buffer and vortex the tube contents thoroughly. The whole (4 tubes) returned to the mortar and rub. 3. After the last washing with Neutralization Buffer extend the centrifugation to 10 min. The STEP II - Lysis supernatant remove carefully. The pellets leave in the tubes. 4. To each of four tubes with pellets add 500 µl of Lysis Buffer. Thoroughly vortex the mixture and transfer to a clean mortar. 5. The mixture rub for about 5 minutes. Transfer the mixture into two test tubes (approximately 2 ml per tube) and add 5 ml of RNase A to each tube. Vortex thoroughly. 6. Incubate the mixture for 10 min at 65 C with shaking (800 rpm). 7. After incubation centrifuge the sample at room temperature for 10 min at rpm. After centrifugation: 7A. Supernatant transfer to new tubes (describe respectively 1 and 2); leave for further analysis (step 8). 7B. Pellets to each pellet add 1000 µl of Lysis Buffer and vortex thoroughly B1. add 5 µl of RNase A and vortex again B2. incubate at 65 C for 10 min with shaking (800 rpm) B3. centrifuge samples after incubation for 10 min at room temperature ( rpm); B4. transfer the supernatant to a new tube (describe respectively 3 and 4) and discard the pellets. 8. Add 15 µl of LP Buffer to supernatants in tubes 1, 2, 3, 4 (from steps 7A and 7B) and incubate 5 min at room temperature. After incubation fill the test tube to a maximum volume using Precipitation Buffer. Vortex and incubate at -20 C for 5 min. 9. Centrifuge the sample at room temperature for 10 min at rpm. Supernatant removed thoroughly. STEP III Binding with magnetic particles 10. Dissolve the pellet by vortexing in 250 µl of Binding Buffer and then add 25 µl of Magnetic Particles. Thoroughly vortex the tube and incubate for 10 min at room temperature. 11. Put the tube in a magnetic stand, and after collecting the magnetic particles discard the supernatant.

8 Caution! In the subsequent steps, after adding each of wash buffer (PreWash Buffer, Wash Buffer, 70% isopropanol) contents of the tube should be gently mixed by inversion until complete separation of magnetic particles from the walls of the tube. Do not vortex test tubes! STEP IV Washing of magnetic particles 12. Magnetic particles suspend in 1 ml of PreWash Buffer. After complete separation of particles from the walls of the tube, incubate for 3 min at room temperature. Put the tube in a magnetic stand and after collecting the magnetic particles discard supernatant. 13. Repeat step 12 using Wash Buffer. Incubate 1 min at room temperature. After collecting the magnetic particles discard supernatant. 14. Magnetic particles suspend in 1 ml of 70% isopropanol. Incubate for 1 min at room temperature. Put the tube in a magnetic stand, and after collecting the magnetic particles discard supernatant. 15. In order to remove residual isopropanol, open the tube and turn the magnetic stand upside down. Leave in this position on paper laboratory for approximately 5 min. at room temperature. STEP V - Elution 16. Magnetic particles suspend by vortexing in 150 l of TE Buffer. Incubate the mixture for 5 min at 65 C and shaking 800 rpm, and then with opened tubes for another 5 min in this same conditions. 17. After incubation, put the tube in the magnetic stand, and after collecting the magnetic particles, transfer the supernatant into a new tube. STEP VI - Precipitation 18. Add 15 µl of LP Buffer to each tube and incubate for 5 min at room temperature. 19. Add 200 µl of 100 % isopropanol (at room temperature), mix carefully the tube contents and centrifuge 20 min at room temperature ( rpm). 20. After centrifugation, discard the supernatant and wash carefully the pellet with 500 µl of frozen 70% ethanol. Centrifuge 3 min at room temperature ( rpm). Carefully remove the supernatant. Leave test tubes for approximately 5 min at room temperature to evaporate residual ethanol. 21. Dissolve the obtained precipitate by vortexing in 50 µl of TE buffer. The obtained DNA can be used directly for PCR.

9 In order to avoid evaporation and crystallization reagents, we recommend securing parafilm bottles of buffers after each isolation. Storage conditions: + 4 C Lot nr Exp. date NOTE! Certain components of buffers can act strongly irritating. When working with all chemicals must comply with health and safety regulations. This product has been designed and manufactured exclusively for research purposes. It has not been tested in the direction of the application for diagnostic purposes and to direct the production of drugs and for the purpose of manufacturing drugs.