SEA-PHAGES | Cluster Specific Primers

Link to this post \| posted 18 Nov, 2016 15:30
svoshell	Ken, That's great that you were able to design the primers in class! I'm planning to have the students search in some of the genomes in the bioinformatics class to see where the nonspecific annealing occurred. A little note about the A4 phage - they often produce fragments with the A2, A3, and A5 primers that are similar in size to the A4 product. If you load 5 ul it's very difficult to tell them apart, but we were able to get good size estimates when we only loaded 2 ul. It confused some of the students initially, but we ended up having some great discussions and I think they learned more than if they'd had only a single fragment show up on the gel! I'm glad to know that the plaque plug version also works! We only tried the lysate procedure this semester. -Stephanie

Link to this post | posted 18 Nov, 2016 15:30

Ken,

That's great that you were able to design the primers in class! I'm planning to have the students search in some of the genomes in the bioinformatics class to see where the nonspecific annealing occurred. A little note about the A4 phage - they often produce fragments with the A2, A3, and A5 primers that are similar in size to the A4 product. If you load 5 ul it's very difficult to tell them apart, but we were able to get good size estimates when we only loaded 2 ul. It confused some of the students initially, but we ended up having some great discussions and I think they learned more than if they'd had only a single fragment show up on the gel!

I'm glad to know that the plaque plug version also works! We only tried the lysate procedure this semester.

-Stephanie

Link to this post \| posted 22 Nov, 2016 00:07
hchunguwfsea	Hi, Ken and Stephanie, Thank you for sharing the PCR experiment protocol. We tried some of the primers designed by Smith et al., they work to certain extent; we also ended up designing primers to distinguish two phages. We are still looking for magic universal primers that can tell cluster-specific phages. As Ken said, plaque works well for PCR experiments. Attached is a protocol summarized by my students during the summer time. Hope it is useful. Happy Thanksgiving. Hui-Min Edited 22 Nov, 2016 00:07 324Kb

Link to this post | posted 22 Nov, 2016 00:07

hchunguwfsea

Hi, Ken and Stephanie,

Thank you for sharing the PCR experiment protocol. We tried some of the primers designed by Smith et al., they work to certain extent; we also ended up designing primers to distinguish two phages. We are still looking for magic universal primers that can tell cluster-specific phages.

As Ken said, plaque works well for PCR experiments. Attached is a protocol summarized by my students during the summer time. Hope it is useful.

Happy Thanksgiving.
Hui-Min

Edited 22 Nov, 2016 00:07

Link to this post \| posted 26 Feb, 2019 20:49
SBS_UNL	Hello, Students last spring used the PCR protocol dated 11-14-17 (found in Protocols of phagesdb), and we accessed the Hatfull primers (link to Google sheets) when ordering primers. I am now getting a message that the page has been deleted. Is there a new link for the primers or ? I would like to use this exercise again this semester – it worked well to reinforce agarose gel electrophoresis as well as for understanding PCR. Cathy Chia Univ. Nebraska-Lincoln Edited 26 Feb, 2019 20:52

Link to this post | posted 26 Feb, 2019 20:49

SBS_UNL

Hello,
Students last spring used the PCR protocol dated 11-14-17 (found in Protocols of phagesdb), and we accessed the Hatfull primers (link to Google sheets) when ordering primers. I am now getting a message that the page has been deleted. Is there a new link for the primers or ?

I would like to use this exercise again this semester – it worked well to reinforce agarose gel electrophoresis as well as for understanding PCR.

Cathy Chia
Univ. Nebraska-Lincoln

Edited 26 Feb, 2019 20:52

Link to this post \| posted 26 Feb, 2019 21:27
debbie	Cathy, The owner of the file got hacked which is where that google sheet resided. I just learned of this today, so I am going to need some time to see if this attached list matches what was posted. In the meantime, here is the list I have. 49Kb

Link to this post \| posted 27 Feb, 2019 00:07
SBS_UNL	Debbie Jacobs-Sera Cathy, The owner of the file got hacked which is where that google sheet resided. I just learned of this today, so I am going to need some time to see if this attached list matches what was posted. In the meantime, here is the list I have. Thank you for the info and spreadsheet. Cathy

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Cluster Specific Primers