DNA extraction DNAdvance

[Strangelove]

During an attempt to isolate DNA from spores and save a days work I started to wonder about the contents of DNAdvance secret ingredients.

isolating DNA from spores does NOT work with this kit, i tried modifying the protocol by adding 0.5mm glass beads together with the lysis buffer proceeded by 3X20 seconds in a bead-beater incubation 20 min. at 60C after the glass beads settled on the bottom the supernatant was pipette out - followed by the rest of the original protocol. - did not work either.

however i would like to know the content of the lysis buffer and the Bind1 buffer and Bind2 buffer (besides beads)
any other recommendations in terms of DNA extraction from stubborn spores (using the kit) are of course appreciated

below is the only description given the manufacture
link to product protocol and details https://www.beckmancoulter.com/wsrpo...000151v001.pdf

Reagent-------------Description----------------Storage Condition Upon Arrival
Lysis Buffer-----------Lysis (clear)-------------------Room Temperature
Proteinase K----------Lyophilized Enzyme----------(-20°C)
Proteinase K Buffer--Proteinase K Buffer (clear)---Room Temperature
Bind1 Buffer----------Binding solution (clear)------Room Temperature
Bind2 Buffer----------Magnetic Solution------------(4°C)
Elution Buffer---------12mM Tris pH 8.0-------------Room Temperature

[Strangelove]

i am looking for information about homemade AMPure/serapure or someone with general knowledge about the underling function of caboxylated beads.

Q1: for instance can carboxylated beads be used for genomic DNA extraction or does the beads need another coating such as streptavidin?

Q2: is there a limit to the size of DNA extracted with carboxylated beads?

Q3: does any know if the DNA affinity for the beads is only depended on PEG and high salt concentrations, or does pH have a effect as well?

as mentioned i have been trying to extract DNA from mycelia and spores with various lysing buffer ect. glass bead disruption prior to AMPure DNA extraction and so on. currently i am able achieve a crud extraction (pore quality low yield) 1-4ng/ul. i have used a set of ITS primers to estimate the quality and reliability of this method but without any success. to be honest i don't know if the eluted samples contain full length gDNA or only fragmented DNA. should probably run a gel..

any help or just a reply would be gladly accepted

[Strangelove]

bump me up

[nucacidhunter]

This kit seems to be designed for extracting DNA from other tissue types. You may try products that are designed for your application.

[han526]

Spore is hard to disrupt. You may try to use steel ball with longer beating for spore disruption. Incubation in FFPE Magpure bind buffer at 60oC helps too.

Originally Posted by Strangelove

During an attempt to isolate DNA from spores and save a days work I started to wonder about the contents of DNAdvance secret ingredients.

isolating DNA from spores does NOT work with this kit, i tried modifying the protocol by adding 0.5mm glass beads together with the lysis buffer proceeded by 3X20 seconds in a bead-beater incubation 20 min. at 60C after the glass beads settled on the bottom the supernatant was pipette out - followed by the rest of the original protocol. - did not work either.

however i would like to know the content of the lysis buffer and the Bind1 buffer and Bind2 buffer (besides beads)
any other recommendations in terms of DNA extraction from stubborn spores (using the kit) are of course appreciated

below is the only description given the manufacture
link to product protocol and details https://www.beckmancoulter.com/wsrpo...000151v001.pdf

Reagent-------------Description----------------Storage Condition Upon Arrival
Lysis Buffer-----------Lysis (clear)-------------------Room Temperature
Proteinase K----------Lyophilized Enzyme----------(-20°C)
Proteinase K Buffer--Proteinase K Buffer (clear)---Room Temperature
Bind1 Buffer----------Binding solution (clear)------Room Temperature
Bind2 Buffer----------Magnetic Solution------------(4°C)
Elution Buffer---------12mM Tris pH 8.0-------------Room Temperature

[Don Stephano]

Check google for US patent no. 5,898,071.

The lysis buffer is likely 0,8 N NaOH + 8% SDS.
I guess bind1 is the PEG-NaCl-buffer and bind2 are the beads (in TE-buffer).

For homemade AmpureBeads: https://ethanomics.wordpress.com/201...pure-xp-beads/

Q1: carboxylated beads could be used for gDNA-extraction, they do not need a further coating.
Q2: There is a lower limit for the size. You can perform size-selection with the beads. It heavily depends on the PEGNA-ratio
Q3: It depends on PEG-NaCl. According to the patent you can use PEG6000-10000 (around 10% w/v) and NaCl >5M. pH and temperature >0<100°C do not matter

Cheers.