We have ordered a MiSeq. I have several questions about ancillary equipment. Saw only one partially related post.
“37 degree forced air incubator”. A list of additional recommended equipment calls for such an incubator. The only place where this seems to apply in a protocol (like the TruSight Tumor Sample Prep) is for the extension-ligation step. The only forced air incubators I find and those which the company recommends are essentially 3 foot tall tissue culture incubators. Unfortunately bench space is sorely limited. My first thought was ‘how about a heat block (with a lid)?’ but a 2nd mention in the protocol specifies “not a heat block” . Drat. Apparently the microplate – filter assembly is too large for heat blocks. Can anyone recommend alternative devices? Alternative protocols?
Library prep in microtubes rather than microplates. The protocol recommends running 16 preps at a time. Some of the steps in the standard protocol, such as using filter plates, would have to be substantially modified, but there are certainly suitable alternatives. Has anyone tried this or do you just use a few wells on the plate?
Centrifugation of microplates at 20 deg. I was told not to cavalierly interpret this as ‘roughly room temp’. The protocols I looked at call for spins up to 10 minutes and forces of up to 2,500 g. The inexpensive plate spinners I was planning on for ‘pulse spins’ do not have timers and do not have temp control. Any recommendations?
Other protocols for cancer sample, such as Qiagen’s “Reader” panels or Archer’s panels seem to have fewer requirements but perhaps they have been less extensively vetted.
As you can tell I am a newbie to NGS, all advice welcome.
“37 degree forced air incubator”. A list of additional recommended equipment calls for such an incubator. The only place where this seems to apply in a protocol (like the TruSight Tumor Sample Prep) is for the extension-ligation step. The only forced air incubators I find and those which the company recommends are essentially 3 foot tall tissue culture incubators. Unfortunately bench space is sorely limited. My first thought was ‘how about a heat block (with a lid)?’ but a 2nd mention in the protocol specifies “not a heat block” . Drat. Apparently the microplate – filter assembly is too large for heat blocks. Can anyone recommend alternative devices? Alternative protocols?
Library prep in microtubes rather than microplates. The protocol recommends running 16 preps at a time. Some of the steps in the standard protocol, such as using filter plates, would have to be substantially modified, but there are certainly suitable alternatives. Has anyone tried this or do you just use a few wells on the plate?
Centrifugation of microplates at 20 deg. I was told not to cavalierly interpret this as ‘roughly room temp’. The protocols I looked at call for spins up to 10 minutes and forces of up to 2,500 g. The inexpensive plate spinners I was planning on for ‘pulse spins’ do not have timers and do not have temp control. Any recommendations?
Other protocols for cancer sample, such as Qiagen’s “Reader” panels or Archer’s panels seem to have fewer requirements but perhaps they have been less extensively vetted.
As you can tell I am a newbie to NGS, all advice welcome.
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