SEA-PHAGES | did you know you can do restriction digests in the microwave?

Link to this post \| posted 05 Dec, 2024 20:22
nic.vega	really! Found here, protocol is: Make reactions as usual, in 1.5 mL tubes (0.2 mL PCR tubes can melt) Arrange tubes so they are off center on the microwave platform Run four cycles of 10 seconds on, 10 seconds off Add loading dye, load & run gel Be amazed Gel image is a HindIII digest of TrixiePhattel genome (with too much DNA per lane, so kind of smear-y). Numbers above lanes are the number of on/off cycles. Reference: Das RH, Ahirwar R, Kumar S, Nahar P. Microwave-mediated enzymatic modifications of DNA. Anal Biochem. 2015 Feb 15;471:26-8. doi: 10.1016/j.ab.2014.11.003. Epub 2014 Nov 13. PMID: 25447491. 88Kb

Link to this post | posted 05 Dec, 2024 20:22

really! Found here, protocol is:

Make reactions as usual, in 1.5 mL tubes (0.2 mL PCR tubes can melt)

Arrange tubes so they are off center on the microwave platform

Run four cycles of 10 seconds on, 10 seconds off

Add loading dye, load & run gel

Be amazed
Gel image is a HindIII digest of TrixiePhattel genome (with too much DNA per lane, so kind of smear-y). Numbers above lanes are the number of on/off cycles.

Reference: Das RH, Ahirwar R, Kumar S, Nahar P. Microwave-mediated enzymatic modifications of DNA. Anal Biochem. 2015 Feb 15;471:26-8. doi: 10.1016/j.ab.2014.11.003. Epub 2014 Nov 13. PMID: 25447491.

Link to this post \| posted 05 Dec, 2024 22:02
viknesh	Hi Nic, It might be useful to gather more side-by-side data for digests done using our standard method vs the microwave method. It might be quicker to use the microwave, but it’s unclear to me if the quality of the data is where we need it to be (I appreciate that the gel might just be a litle smeary). At the moment, we are beginning to look for phage DNA modifications, which can be gleaned by looking for inconsistencies in the expected digest pattern (i.e. a virtual digest based on the DNA sequence) vs pattern observed when the actual digest is run on a gel. If it is tricky to get good digests and gels using the microwave method, then that’ll impact the utility of the data for that purpose. The TrixiePhatell gel from a regular digest can be viewed here. We’re always excited to hear of new ways of doing our collective research. As we learn of new strategies though, the SEA team usually invests time to understand the implications of the new technique for our collective work before we incorporate anything into our regular workflow. This is so we can continue to leverage the large datasets that are generated by faculty and students from across the program to glean new insights. If some of that data is generated differently, it can impact our interpretation. So if you are willing to gather some data to be reviewed, perhaps over the next semester, please do share. Best, Vic

Link to this post | posted 05 Dec, 2024 22:02

viknesh

Hi Nic,

It might be useful to gather more side-by-side data for digests done using our standard method vs the microwave method. It might be quicker to use the microwave, but it’s unclear to me if the quality of the data is where we need it to be (I appreciate that the gel might just be a litle smeary). At the moment, we are beginning to look for phage DNA modifications, which can be gleaned by looking for inconsistencies in the expected digest pattern (i.e. a virtual digest based on the DNA sequence) vs pattern observed when the actual digest is run on a gel. If it is tricky to get good digests and gels using the microwave method, then that’ll impact the utility of the data for that purpose. The TrixiePhatell gel from a regular digest can be viewed here.

We’re always excited to hear of new ways of doing our collective research. As we learn of new strategies though, the SEA team usually invests time to understand the implications of the new technique for our collective work before we incorporate anything into our regular workflow. This is so we can continue to leverage the large datasets that are generated by faculty and students from across the program to glean new insights. If some of that data is generated differently, it can impact our interpretation. So if you are willing to gather some data to be reviewed, perhaps over the next semester, please do share.

Best,
Vic

Link to this post \| posted 05 Dec, 2024 22:22
nic.vega	Sure, we can give it a try! I'll plan on having some of my folks try microwave vs traditional digests with some commonly-used enzymes and see how they compare.

Link to this post \| posted 10 Dec, 2024 18:11
nic.vega	We did some side-by-side reactions on TrixiePhattel; results attached. The gel was much better this time - it turns out the reason that the first gel was blurry is that the 10X TBE in the teaching lab expired in 2012. 154Kb

Link to this post \| posted 10 Dec, 2024 19:21
viknesh	nic.vega We did some side-by-side reactions on TrixiePhattel; results attached. The gel was much better this time - it turns out the reason that the first gel was blurry is that the 10X TBE in the teaching lab expired in 2012. Thanks for sharing this – very nice gel. If your student is willing to do it one more time with a third setup: Regular 1 hr; Regular 1 min, MWV 1 min, that'd be helpful. Even in our regular setup (no microwave),I think it is likely that the majority of DNA is restricted within the first minute, which might potentially give us the same results as the MWV reaction. Thanks, Nic. Vic

Link to this post | posted 10 Dec, 2024 19:21

viknesh

nic.vega
We did some side-by-side reactions on TrixiePhattel; results attached. The gel was much better this time - it turns out the reason that the first gel was blurry is that the 10X TBE in the teaching lab expired in 2012.

Thanks for sharing this – very nice gel. If your student is willing to do it one more time with a third setup: Regular 1 hr; Regular 1 min, MWV 1 min, that'd be helpful. Even in our regular setup (no microwave),I think it is likely that the majority of DNA is restricted within the first minute, which might potentially give us the same results as the MWV reaction.

Thanks, Nic.
Vic

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did you know you can do restriction digests in the microwave?