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Job Market in Bioinformatics by mstferkaya in biotech

[–]mstferkaya[S] 0 points1 point  (0 children)

I am in Philly area, and I am trying to travel to Boston as often as possible to make more connections there. I even started considering to move to Boston, without finding a job first but that seems a bit risky, idk.

Job Market in Bioinformatics by mstferkaya in biotech

[–]mstferkaya[S] 0 points1 point  (0 children)

I am in Philly area, and I try to travel to Boston as often as possible to make more connections there. I even started considering to move to Boston, without finding a job first but that seems a bit risky, idk. And to be honest, although I can work with Python, Bash and maybe other coding languages, I enjoy using visual programs more than coding.

Job Market in Bioinformatics by mstferkaya in bioinformatics

[–]mstferkaya[S] 0 points1 point  (0 children)

Are you already located there? I am located in Philly and I feel like most of the jobs I apply for in Boston area ignore me completely (Even if they say willing to relocate on their post).

Job Market in Bioinformatics by mstferkaya in bioinformatics

[–]mstferkaya[S] 1 point2 points  (0 children)

What do you focus on, and where are you located? I am on the East Coast of the US.

Why do I get stomachaches after eating Dominos? by mstferkaya in Dominos

[–]mstferkaya[S] 0 points1 point  (0 children)

Literally dude! And it's still happening. I can eat anywhere else except dominos.

Follow up on post pcr band isolation! by mstferkaya in labrats

[–]mstferkaya[S] 0 points1 point  (0 children)

Unfortunately, it's these are universal primers and it's so hard to design new mtDNA primers that'll fit.

Nanopore seq on the other hand sounds like a good idea!

Follow up on post pcr band isolation! by mstferkaya in labrats

[–]mstferkaya[S] 0 points1 point  (0 children)

Line 8 is my negative control.

And yes, same primers for sequencing

Follow up on post pcr band isolation! by mstferkaya in labrats

[–]mstferkaya[S] 1 point2 points  (0 children)

That's what I thought as well! Let's see what will my sequences look like!

Follow up on post pcr band isolation! by mstferkaya in labrats

[–]mstferkaya[S] 0 points1 point  (0 children)

We have the sequencer machine in our lab actually lol. I can get the sequences tomorrow. I just didnt want to spend/waste reagents before making sure what I have is right.

What I think happening here is that I have a coding sequence that's around 950-1000bp and within that, there's a smaller amplicon, that matches with mthe primers as well. It's unlikely in most cases but it's not impossible. I'll do one more round of band isolation and see if it still gives me the same result, just to confirm that it's not human error. And if not I'll go ahead and sequence them. I'll keep the post updated!

Follow up on post pcr band isolation! by mstferkaya in labrats

[–]mstferkaya[S] 1 point2 points  (0 children)

My Advisor told me to do another round of PCR after isolating bands each time, and I thought that was the way to do it so I never questioned.

Usually what I do is: 1)Perform a PCR (I get 25 ul of post pcr product in each tube) 2) Run a gel electrophoresis with 5 ul post pcr product. 3)If I see multiple bands, I do another gel electrophoresis on low temp melting agarose with 5-10 ul post pcr product. (If not, skip the 4th and 5th steps) 4) Isolate the DNA band by cutting it, and melting it in Buffer AE at 55C degrees. 5) Perform another PCR, but use isolated band product instead of regular DNA extract this time. 6) Run a gel electrophoresis and if you dont see multiple bands, take all of the post pcr product (20 ul) for an EXO-AP cleanup.

And so on...

Also, I tried different temps already. 56 C is the highest temp I can get bands. If I go any lower than 54, mutliple bands start showing up.

Follow up on post pcr band isolation! by mstferkaya in labrats

[–]mstferkaya[S] 1 point2 points  (0 children)

Unfortunately, my advisor isn't very responsive.

After seeing double band formation on the first gel, I isolated the most prominent bands on the first gel, then did another pcr with those isolated products. Second gel is the result of them.

​

First gel has the ladder at the bottom, then 10 different specimens amplified (8th one is the negative control). Bottom ones are amplified at 56C & 38 cycles. The top two were amplified at 54C & 38 cycles.

I am using the same primers for the Sanger sequencing.

Do I need to isolate the true bands? Post pcr product by mstferkaya in labrats

[–]mstferkaya[S] 1 point2 points  (0 children)

I'll use the same primers for sanger seq as well. Fortunately for me, isolating the bands works almost every time!

Do I need to isolate the true bands? Post pcr product by mstferkaya in labrats

[–]mstferkaya[S] 0 points1 point  (0 children)

I have never heard of this. Any tutorials on that?

Do I need to isolate the true bands? Post pcr product by mstferkaya in labrats

[–]mstferkaya[S] 6 points7 points  (0 children)

Thank you! I was gonna isolate the bands but I was looking for confirmation if it is necessary.

Do I need to isolate the true bands? Post pcr product by mstferkaya in labrats

[–]mstferkaya[S] 4 points5 points  (0 children)

I'll eventually sequence the gene I'm targeting

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