BOMB protocol #7.1 gDNA extraction

Viewing 15 posts - 1 through 15 (of 16 total)
  • Author
    Posts
  • #1610
    PStepper
    Keymaster

      Hi everyone,

      this is the main discussion space for BOMB protocol #7.1 gDNA extraction.
      Here you can post all your questions and feedback!

      #1611
      PStepper
      Keymaster

        reserved space

        #2083
        AKSchilling
        Participant

          Hi,

          thanks for your helpful protocols. Do you have or working at a protocol for gDNA extraction for insects?

          #2085
          Tomek
          Keymaster

            Dear AKSchiling,

            We never tried extracting NA from insects. I suppose this should not be too difficult to optimize. Probably you could follow the “Tissue extraction” protocols, where you would release the NA with ProtK treatment and then GITC buffer for cell lysis and NA capture.

            Cheers,

            Tomek

            #2089
            TimH
            Keymaster

              Hi AK Schilling – sorry for the slow reply – in case you are still wondering whether to give it a go: yes, people in my Department have tried BOMB on insects, following the Tissue extraction protocol as suggested by Tomek. I don’t think the chitin exoskeleton was broken down, but DNA was extracted all the same.

              Cheers,

              Tim

              #8903
              Audrey McDonald
              Participant

                Hi, thanks so much for all the information and protocols!

                Do you have any recommendations for modifying the gDNA extraction protocol for plant tissue extractions?

                #8922
                Phil Oberacker
                Keymaster

                  Hey Audrey,

                  there is a protocol for plant tissue TNA extraction available (#6.4). The protocol also contains a gDNA version, as you simply do an RNase digest after lysis.

                  Cheers
                  Phil

                  #8926
                  Jon Sanders
                  Participant

                    In your paper, you mention the possibility of substituting in commercially prepared magnetic particles (such as these). We would like to try these out. How exactly are you preparing them? Presumably they are diluted somehow?

                    #8927
                    TimH
                    Keymaster

                      Hi Jon – to prepare those beads for gDNA, RNA and TNA extraction take 1 mL of thoroughly resuspended bead mixture and place on a magnetic. Once beads have settled, remove the storage buffer (it contains sodium azide) and resuspend in 1 mL of TE buffer off the magnetic. Repeat process at least twice more and then resuspend in TE. We do this into 50 mL of TE – seems to be a good concentration of beads, however this could likely be altered without making much difference.

                      Cheers, Tim

                      #8948
                      Nuria
                      Participant

                        Hi,

                        Thank you for sharing these efficient protocols. I am doing gDNA extraction from gonads (ovaries and testis) with your protocol (6.3). However, I am getting low 260/230 ratios. What would you recommend?

                        Cheers,

                        #8950
                        admin
                        Keymaster

                          Ahoy,

                          As the low 260/230 might be the result of GITC being carried over into to the final elution the first thing you should try is performing one or two additional 80% EtOH wash steps.

                          Cheers

                          Tim M

                          #9513
                          Jennifer Kuehl
                          Participant

                            Hello

                            I am also getting very low 260/230 ratios.  I tried up to eight 80% ethanol washes, the ratios  improve with more washing but  they are still low ~.6 for gram positive bacteria and 1.7 for gram negative.  I am considering  a larger volumes of 70% ethanol (600ul) for the wash steps or perhaps a secondary bead clean-up.  Any recommendations?

                            #9522
                            admin
                            Keymaster

                              Not really, at least nothing you haven’t suggested yourself. I frequently use 70% EtOH for my washes so that should work but if it will solve you issue I cannot tell.  Are you making sure there is no liquid transferred from wash to wash inside the tube lid? Cheers

                              #9712
                              Emilly
                              Participant

                                Hello!

                                I was reading your protocol of gDNA extraction with beads and a got confused with ratio mentioned 2:3:4. How is it used with blood sample and without TE buffer at beggining? It’s 140ul of blood and 20ul magnetic bead?

                                Best regards.

                                #9717
                                TimH
                                Keymaster

                                  Hi Emily,

                                  Blood does not really work that well with the 2:3:4 ratio, as there is a lot of liquid in there and (for humans/mammals) not many nucleated cells. You are probably better off going with a 2-step ’tissue’ protocol (#6.3), where you first protK treat in TNES, and then add in 1.5X GITC. From this point on it is basically 2:3:4.

                                  Best wishes, Tim H

                                   

                                Viewing 15 posts - 1 through 15 (of 16 total)
                                • You must be logged in to reply to this topic.