Hybridization and background problems - uneven signal and background noice (Apr/24/2008 )
Hi everyone,
I read from other posts regarding to hybridization and background problems, but i am still not sure about some details.
1. Someone mentioned about extend the time for Pre-hybrid to several hours (i think its the step when you block your slide with BSA or Goat serum), my protocols says that i should pre-heat my prehybrid buffer at 42 degree for about 15 mins which i did, then incubate at 42 degree for at least a hour after you put the slide in the buffer. SO my first question is 90 mins of pre-hybrid too long or too short?
Also i put my jar (pre-hybrid buffer + slide) on a rotor, and rotate at very slow speed during the 90 mins incubation, is that a good idea?
2. I noticed that pretty much all of my slides (after scan) has a trend of decreasing in signal strength, i loaded the hybridization mixture (buffer and labeled cDNA) from one side of the cover slip which is elevated for about 15 degree, right after i heated my mixture for 5 mins at 95 degree. and the probe mixture will slowly migrate to the bottom side of the array under the cover slip.
So, what i noticed is the side where i load the probe always has good signal strength and hybridization, but it kinda gets weaker and weaker further, and sometime no signal or hybridization at the end of the array. SO, i wanted to pre-heat my array at 80~85 degree for few minutes prior to loading the probe, is that a good idea?
Also, is it a good idea to increase the temperature for Hybridization? I am currently using 42 degree.
3. I read from other old posts saying that dry your slide inside a falcon tube will reduce that background problem, because i used a mini-array centrifuge and always get get uneven result (it's like split right in the middle of the array. left portion of the slide looks good while the right portion looks terrible), so my question is that other is drying the slide in a falcon tube the best way to go?
4. i talked to several people about the post-hybrid wash steps. Some told me to wash longer and more times which can reduce the background, and some say wash with shorter time. SO which one is better i washed with three different wash butter from high salt concentration with SDS to low salt concentration without SDS.
FYI: i use Tigr Array with foramine hybridization buffer with axon 4000b scanner, and i pre-hybrid my slide with BSA
Thank You Very Much.
Anyone attending 2008 ASM general meeting?
Thanks
Frank
[quote name='frank502lin' date='Apr 24 2008, 04:58 PM' post='133773']
Hi there,
Sorry for not seeing your post earlier, but I hope you've solved the problem now. However, just in case this is the protocol I follow as I am using TIGR arrays too. ftp://ftp.jcvi.org/pub/data/PFGRC/pdf_fil...tocols/M008.pdf
1. I have had a lot of problems with background and especially on the Cy3 channel. I only pre-hyb for 1 hour in pre heated buffer at 42 degrees; so that should be fine but I don't rotate mine I leave it in a coplin jar in the dark incubator. I wash my slides in slide racks and glass troughs filled to brim with the water, then Isopropanol. Wash quite vigorously but don't let the slides come out of the liquid. Always keep them in liquid as they dry very quickly.
2. When loading the hyb mix from the top, add any excess to the bottom of the cover slip to allow even binding. If possible retain some hyb mix to do this. This works for me.
42 degree should be fine but if you want to increase the stringency then I have used 47degree which also works well. You can work out your binding melting temps using this equation: The net effect of these factors on the Tm of a DNA–DNA hybridisation can be expressed using the following equation (Meinkoth and Wahl, 1984):
Long probe
81.5 + 16.6(log10 M) + 0.41*(% GC) - 0.61*(% form) - 500 / Length in bp
(where M is the molarity of Na+ (set at 0.75 M)
and % form is the percentage of formamide (set to 50%)) Meinkoth and Wahl (1984)
Eg: %GC=49, %form=50, n is the length of the shortest duplex = 70bp
0.83MHyb solution: Tm=81.5+16.6*log 0.83M+20.09-30.5-7.1= 62.65°C
no formamide
0.33MLow stringency wash: Tm=81.5+16.6*log 0.33M+20.09-7.1= 86.5°C etc... for each buffer
then, Given that a 1% mismatch corresponds to a 1°C drop in temperature, a hyb temp of 42°C allows a 21% mismatch over 70bp in the hyb solution (62.65°C – 42°C = 20.65%).
3 I put my arrays straight into a falcon tube and centrifuge for 5 minutes at 1,500xg. Works for me.
4. Follow the TIGR protocol. Works for me.
Hope this helps even though it's late.
How are things going now and what does your Cy3 channel look like compared to Cy5 ?
Thanks
LadyGiardia