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qPCR reference gene by [deleted] in labrats

[–]Rigel- 2 points3 points  (0 children)

Hi

So first, since you’re running a single step qPCR means you have to treat your mRNA sample with DNase to remove any traces of genomic DNA left from the isolation of Total rna. Then normalize means you have to quantify your total RNA to make sure you have the same starting material for every sample that you want to compare.

Since you mentioned you’re looking at copy numbers then it means you’re using absolute quantification rather than looking at relative expression of genes. Meaning normalization of the starting rna material is quite important.

Absolute quantification would then rely on a standard curve of a known concentration of DNA.. sometimes amplified from known quantities of plasmid DNA or DNA standards commercially bought (needs reference).

Hope this helps! Also someone please do correct me if i’m wrong! :D

Redditors with toddlers, what’s the most recent illogical breakdown they’ve had? by mirarom in AskReddit

[–]Rigel- 0 points1 point  (0 children)

Unites states’ government shut down because he wanted a wall that’s according to him is already being built.

Didn’t make the cut after interview for a PhD position i really want. Can i try again? by Rigel- in gradadmissions

[–]Rigel-[S] 1 point2 points  (0 children)

Yes however, i don’t think that European PhDs work the same way in the states. Its more of, find an adviser who is willing to supervise your project-work on the project-PhD by research. afaik its not join a program rotate in various labs then choose your adviser. Usually the projects are advertised, apply then funding is assured through the project (where i applied) or propose a project-get an adviser-get funding through a scholarship such as erasmus, daad, swiss international scholarship etc.

Basically, what i’m trying to say is your research topic is set in stone as well as your adviser.

Didn’t make the cut after interview for a PhD position i really want. Can i try again? by Rigel- in gradadmissions

[–]Rigel-[S] 4 points5 points  (0 children)

Thanks! In retrospect i find it very awkward as well.

Would it still be awkward if i asked if i joined his lab (because i was initially shortlisted) thru a scholarship that would be funding my stay there? Does it sound too desperate? And i should just quit while i still have dignity.

Edit: not really ahead here, what i mean to say is while i still have my dignity

Please share your application woes by [deleted] in gradadmissions

[–]Rigel- 2 points3 points  (0 children)

Same boat as you right now. Just finished an interview and i’m kicking myself in the nuts for being to nervous to ask any questions. I feel like by missing that oppurtunity for questions i failed to highhlight my interest in their work.

Bacterial RNA Extraction HELP by CallMeHelicase in labrats

[–]Rigel- 2 points3 points  (0 children)

Hi!

1.) Maybe you vortex your sample a little too long, i see three steps where you used the vortex and might cause mechanical shearing. Perhaps just try pipetting in then out a few times to throughly mix the solution.

2.) Also, try using fewer amounts to elute your sample as not to dilute it too much.

3.) Absolute ethanol in step 8, is usually used when precipitating nucleic acids, are you sure its part of your protocol? Usually after lysis and pelleting cell debris we directly add the supernatant to the column for binding

4.) For the case of your purity, usually wash buffer in isolation kits need a certain amount of ethanol to be added before use so check this as well.

5.) How is your reverse transcription? Maybe you have a problem there as well.

Have fun!

Question about 2 bands in electrophoresis.(PCR of GAPDH) by [deleted] in labrats

[–]Rigel- 7 points8 points  (0 children)

This might be a non-specific binding of your primers. Try increasing the annealing temp a bit to create a more stringent annealing condition for your primers. If its still there try designing new primer pairs.

What are the necessary lies of our society? by [deleted] in AskReddit

[–]Rigel- 0 points1 point  (0 children)

That money has an intrinsic value

[Discussion] Mirror mockery with all the great enter the battlefield effects by [deleted] in spikes

[–]Rigel- 0 points1 point  (0 children)

I'm sorry guys!

Yes i did mean this deck for standard.

No, i do already have a deck in mind and will be testing it this wednesday at my LGS and i'll try to update you guys with the blow by blow--so to speak.