3 Replies Latest reply on Feb 1, 2019 11:07 AM by agrafsky

    RevoverEase protocol versionD questions

    sayano

      Hi Tech support team, We have received some questions regarding the new protocol of RevoverEase. Could you please tell us the answer?

      In P5,step 25, “Immediately spin the conical tube containing the dialysis tube for 5–6 seconds at 500–1000 × g. Do not spin longer than 6 seconds as this may cause the membrane to rupture”.

      Q1, Should we count 5-6 second when the centrifuge reach the appropriate speed which is 500-1000 x g, not from starting the spinning? I think usually  it should be counted after reaching the desired speed, but I would like to confirm.

      Q2, How immediately should we start spinning after dialysis? What could be the negative impact if we don’t spin the tube immediately after dialysis?

       

      Thank you for your support in advance.

      Best regards,

      Sayano Kinjo

        • Re: RevoverEase protocol versionD questions
          agrafsky

          Ordinarily, most centrifuges will begin application of the necessary force within less than a second of their starting, so it would be fine to start counting the 5-6 seconds as soon as the centrifugation begins. Obviously, if the particular centrifuge requires several seconds to reach the point where it is generating 500 x g, then it would be best to wait until it reaches the speed at which this force is generated.

           

          The main reason for centrifuging immediately is that the genomic DNA should not be allowed to become dehydrated and the conical tube cannot be sealed until the dialysis cap is replaced with the storage cap. Once the tube is sealed, then the gDNA may be stored.

          1 person found this helpful
            • Re: RevoverEase protocol versionD questions
              sayano

              Hi Albert,

              Thank you for your helpful reply. I have received some additional questions.

               

              in the protocol, P4, 18 step, it is stated,

              "After the proteinase K digestion step is complete, add 300 μl of
              TE buffer to the DNA sample and stir the sample using a wide-bore pipet tip."

               

              The customer is asking whether  they can decrease the volume of this TE buffer from 300 ul to, for example 150 ul and increase the dialysis time longer than 5 days (such as 8 days) .

               

              The reason why they ask this question is that, they want to increase the concentration of gDNA after dialysis. In order to do this, they think decreasing the TE buffer volume could be a good way.

              I doubt if our R&D has ever tested such condition, but if you have any information, or thought,  could you please share?

                • Re: RevoverEase protocol versionD questions
                  agrafsky

                  Hello Sayano,

                  The conditions described in the product manual were applied so as to match the packaging efficiency of the gDNA isolated with the new dialysis columns with that of the old dialysis cups as closely as possible.

                  Obviously, customers can try lowering the TE Buffer and increasing the dialysis time, depending on their downstream application. However, in our hands, lower volumes of buffer resulted in gDNA solutions that were too viscous to handle effectively. Considering those results, it would probably be best if the customer did NOT apply the changes they proposed.