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shawn6022

Member Since 26 Nov 2012
Offline Last Active Jun 08 2013 06:45 PM

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Group Active Members
Active Posts 13
Profile Views 284
Member Title member
Age 32 years old
Birthday December 30, 1980
Gender
Male

About me

Real Name
Shawn
Institution
ETSU
My research interests
mycobacterium spp, dictyostelium discoideum, and pseudomonas spp.

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2xzwei
16 Apr 2013 - 10:44

Posts I've Made

In Topic: Problems with crossover PCR

15 April 2013 - 03:28 PM

I thought about gel extracting it the 2.5 kb band but I was afraid of losing a good bit of the SOEing product and with the low amount already there I was afraid of losing all of it. I guess it's worth a shot anyway. I can try to gel extract it and subclone it.
I guess I'm more concerned with what could cause the band at 1.3 kb? It looks like each of the fragments are amplifying without annealing to one another..it kind of looks like there is a space in the middle where there are two bands...maybe I'm just seeing things...

In Topic: problems with plasmid miniprep

15 April 2013 - 12:26 PM

Hey, I just wanted to let you know that your method of adding the acetate quickly after the lysis step worked. I was able to isolate a beautiful band of both the insert and the vector after the restriction digest. Thanks again!

In Topic: problems with plasmid miniprep

11 April 2013 - 06:38 PM

Thanks and I apologize for the confusion. I will remake the NaOH tomorrow and try what you said about adding the acetate right after the lysis step. I've noticed that different protocols have various times to keep the cells in the lysis solution before adding the acetate..I will try it and let you know how it goes...once again I apologize for the confusion..

In Topic: problems with plasmid miniprep

10 April 2013 - 06:27 PM

Ok, after doing some calculations the final concentration of the NaOH is 0.2 M. I make the solution with 4.4 mL ddH2O, 500 uL 10 % SDS, and 100 uL 10 M NaOH...the final concentration is 1% SDS and 0.2 M NaOH...

In Topic: problems with plasmid miniprep

08 April 2013 - 02:34 PM

Thanks. i will try the lower molar NaOH and use 1% SDS instead of 10%. I usually use 2.5 volumes EtOH but I was looking at a different protocol when I typed this out. I hope this works...I manage to get a good amount of genomic DNA when doing this...do you think that the higher molar concentration of NaOH is causing that?

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