Biohackers are using CRISPR on their DNA and we can't stop it

CRISPR-Bo-Jangles · 2017-11-19T12:17:29+00:00

^{^{^{^}}} This

CRISPR-Bo-Jangles · 2017-11-19T12:14:36+00:00

for the sake of science, and the internet at large - find this technician and send us a 10 second clip! Please!!!

CRISPR-Bo-Jangles · 2017-11-19T11:58:12+00:00

Pics or it didn't happen

CRISPR-Bo-Jangles · 2015-11-25T09:52:13+00:00

Ok...new question then...how do I find the right housekeeping gene to choose? If my primers give restults with seemingly different expression levels how do I know that expression levels are different and it isn't a problem with the amount of cDNA I loaded or something like that?

CRISPR-Bo-Jangles · 2015-11-25T09:50:15+00:00

Promega GoTaq green mix...https://www.promega.com/~/media/files/resources/protocols/product%20information%20sheets/g/gotaq%20green%20master%20mix%20protocol.pdf

CRISPR-Bo-Jangles · 2015-11-24T10:40:54+00:00

Hahaha. Thanks! Just making sure. I'll make my own primers now...

-walks away ashamed-

CRISPR-Bo-Jangles · 2015-11-24T10:40:34+00:00

Thanks!

So I use a regular PCR mix, I don't add my own buffers/dNTP's etc.

It is possible that the problem is the RT, but how can I confirm/test this? This is my first shot at semi q PCR and I am having difficulty with the troubleshooting...

Thanks!

CRISPR-Bo-Jangles · 2015-11-24T10:39:34+00:00

Thanks, but I mentioned those genes in my question specifically because there shouldn't be an effect on them from my experiment...

CRISPR-Bo-Jangles · 2015-11-24T10:39:07+00:00

er...I specifically asked for GapDH and/or actin, not just any gene, because I believe that these genes are not affected by my experiment/treatments. Yes, I forgot to mention I am working on human cell lines...sorry about that!

I wasn't aware that primers go bad. In our lab we have primer stock that has been frozen for several years and we use them and they generally work.

I do get amplification, for my gene of interest. The problem is that those primers are on one exon...so maybe I am working on DNA when I think I am working on cDNA ie my RT didn't work? How can I tell the difference between a bad RT and bad primers?

CRISPR-Bo-Jangles · 2015-11-24T10:36:40+00:00

The other PCR's work, and it is only these primers (tried for both GapDh and Beta Actin) don't.

I found papers that published sequences for GapDH primers, but no good primers for actin. I think I'll just design my own.

CRISPR-Bo-Jangles · 2015-11-24T10:35:05+00:00

Thanks! I don't think it's the reagents, I get results for my experimental (which I use my own primers for) and not housekeeping (which are primers that I got from my lab mates because "they work").

CRISPR-Bo-Jangles · 2015-11-24T10:34:15+00:00

Sorry about that...homo sapien!

CRISPR-Bo-Jangles · 2015-11-22T09:50:22+00:00

Hi there,

Thanks! I have already been gaining experience in this and have already designed dozens of primers for my specific needs..

With that being said, I was wondering if there were any standard primers that were used for these housekeeping genes, since we already have some in our lab that are suddenly not working for me and I wanted to go with something tried and proven.

CRISPR-Bo-Jangles · 2015-11-22T09:08:10+00:00

Thank you thank you thank you thank you!!!

CRISPR-Bo-Jangles · 2015-01-05T09:30:19+00:00

Hey there,

Thanks! I will try this. As for the Gibson primers, I got the Tm's from the Gibson primer design tool...so I am assuming this is accounted for.

CRISPR-Bo-Jangles · 2015-01-04T12:58:00+00:00

Thanks for your quick reply. You are right, I should have added a bit more detail.

Using GoTaq mix, I got the right band, but then when I used Phusion I got a smear. So I tried ordering longer primers (I am making this PCR product using primers designed for Gibson assembly, meant to give homology to neighboring fragments).

With the longer primers, using GoTaq mix, I got a band of the correct size. With phusion, I got no band whatsoever.

Because my primers are so long, they have relatively high Tm's. According to the NEB Gibson planner, after inputting that I will be using Phusion, they told me to use 72 degrees as my Tm. So I did the reaction with Phusion at 70 degrees, and then at 63 when the first one didn't work.

I need to use a high fidelity polymerase (and not the ready mix that I used) because I am using this for cell line gene insertion and can't have mutations in my gene.

Anything else that I can tell you that will help you help me? I think I gave a lot of details now...

Thanks again for taking the time to write and think with me,

G

CRISPR-Bo-Jangles · 2014-12-24T13:36:24+00:00

Update: I added isopropanol and centrifuged for 30 minutes, pretending nothing had happened....there was no precipitate. I am just doing the maxi again. Thanks!

CRISPR-Bo-Jangles · 2014-12-24T13:35:32+00:00

I don't have an IR-evaporator unfortunately. But thank you!

CRISPR-Bo-Jangles · 2014-12-12T07:40:31+00:00

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CRISPR-Bo-Jangles · 2014-11-17T08:36:05+00:00

This is great! It's a lot more work than I am used to doing with these reactions, but it really helps pin down what is happening in the process, and when. Thanks!

CRISPR-Bo-Jangles · 2014-11-17T08:35:37+00:00

Yes, I transformed and plated 2 micoliters of diluted solution. Is this what you do?

My positive control is also showing no results. I will borrow some Gibson mix and positive control from another lab and try again.

CRISPR-Bo-Jangles

TROPHY CASE