5 replies to this topic

[vetticus3]

Hello,
I've just realised that I added 10X more salmon sperm to my ChIP than I was supposed too... ... i just can't brain today.

Any idea on what will happen?
Any ideas on how i can fix this?

V

[KPDE]

vetticus3, on Oct 11 2009, 07:27 PM, said:

Hello,
I've just realised that I added 10X more salmon sperm to my ChIP than I was supposed too... ... i just can't brain today.

Any idea on what will happen?
Any ideas on how i can fix this?

V

In our plate based assay, increasing the salon sperm DNA beyond a certain point increased background. I don't know if it would do the same if you're using beads. In any case, there's not much to be lost by going through and completing the assay.

[vetticus3]

i pushed ahead anyway... but i've got my cells up and growing as an insurance plan. PCR in a few hours... we'll see. somedays, i swear, i don't see the decimal point.

hypothetically speaking, is it possible to "over" reverse cross link? say, if i left it for 18 hours ... i don't want to spend 14 hours days at work.

V

[KPDE]

vetticus3, on Oct 12 2009, 11:59 PM, said:

i pushed ahead anyway... but i've got my cells up and growing as an insurance plan. PCR in a few hours... we'll see. somedays, i swear, i don't see the decimal point.

hypothetically speaking, is it possible to "over" reverse cross link? say, if i left it for 18 hours ... i don't want to spend 14 hours days at work.

V

You can definitely overcrosslink. It can make shearing impossible if you crosslink long enough and it can mask epitopes as well (especially if they depend on lysine).

[jiro_killua]

KPDE, on Oct 15 2009, 08:25 PM, said:

vetticus3, on Oct 12 2009, 11:59 PM, said:

i pushed ahead anyway... but i've got my cells up and growing as an insurance plan. PCR in a few hours... we'll see. somedays, i swear, i don't see the decimal point.

hypothetically speaking, is it possible to "over" reverse cross link? say, if i left it for 18 hours ... i don't want to spend 14 hours days at work.

V

You can definitely overcrosslink. It can make shearing impossible if you crosslink long enough and it can mask epitopes as well (especially if they depend on lysine).

I thought he said over "reverse" crosslink

and regarding that, I think reverse cross link time can be quite flexible, and usually put at 65C with 10ul 5M NaCl overnight

[vetticus3]

haha, i have found my answer...
too much salmon sperm makes a bit more background, but nothing to get fussed over.

i didn't try to over reverse crosslink... i wussed out and waited for the 6 hours.

V