[martona]

Hi everybody,

Is anybody familiar with the fragmentation buffer below? I need to know the proportion of the below. This is what I 've found in a paper. I am trying to get fragments of about 200 bp excluding the adapters for SOLID sequencing. mRNA was fragmented at 82 degrees for 2 minutes..
Does anybody have any ideas ?

40mM Tris-acetate
100 mM Potassium acetate
31.5 mM Magnesium Acetate
pH 8.1

Did you try this buffer? I would like to use it

[scooter]

The frag buffer will have a 1x concentration of ~10 mM Zn2+. This is pretty high so I think any amount of EDTA that might get co-precipitated will be very low and probably not worth worrying about.

[Nath1606]

Thank you very much, I was hoping you would say that! Thank you. As you said it would get rid of 'most' of the EDTA, do you think I will need to add more frag buffer when I repeat this fragmentation to compensate for small amounts of EDTA remaining which may affect fragmentation?

[scooter]

Precipitation should work fine to remove most of the EDTA from the RNA. Even a G-25 or G-50 spin column would work as a good buffer exchange.

[Nath1606]

Apologies for intruding on this topic, but I need your advice. I accidentally added the STOP solution instead of the fragmentation buffer to fragment... Any ideas if ethanol precipitation will suffice in getting rid of the EDTA so I can then add frag buffer and start again or does EDTA precipitate too and therefore would I need to phenol extract prior to etoh precipitation? Your knowledge I'd much appreciated.

[scooter]

In my experience, fragmentation of RNA is highly sample specific. That is, plant RNA will behave differently than mammalian RNA, which is different than bacterial RNA, etc.... Also, there are typically differences in RNA purified by different methods. So it is usually best to perform some pilot experiments to determine the best time and temp to fragment your particular RNA samples.

Any of the fragmentation buffers mentioned in this thread will likely work quite well. The main point is to have some sort of metal ion (Mg2+, Zn2+, etc.....). There are some slight differences in efficiency of fragmentation that depends on metal ion concentration or type, but this is not the strongest variable. Sample type seems to have the highest degree of variance, so you will want to simply run your RNA with a few different conditions to see what produces the best size range.

Also, in my experience the biggest 'problem' with fragmentation is making a LOT of very small products (say less than 20 nt) that are normally quite difficult to detect by typical methods like PAGE since they tend not to stain very well with SYBR gold or EtBr due to their small size. So some sort of clean up step is recommended to try and remove this small material. The best clean up is to gel purify the RNA, but that is not so convenient.

[Tulay Bayram]

mRNA Fragmentation buffer

Hi everybody,

Is anybody familiar with the fragmentation buffer below? I need to know the proportion of the below. This is what I 've found in a paper. I am trying to get fragments of about 200 bp excluding the adapters for SOLID sequencing. mRNA was fragmented at 82 degrees for 2 minutes..
Does anybody have any ideas ?

40mM Tris-acetate
100 mM Potassium acetate
31.5 mM Magnesium Acetate
pH 8.1

[silin284]

Hi Xile

120 is the after PCR size. the SR pcr primers are 58 nt and 34. PE pcr primers are 58 and 61.

both SR and PE adapters are around 30 nt. So after ligation, only 60 bp were added to the ds cDNA, not 120.

[xile]

Hi silin284, as I know that the PE 2 adapters are around 120.

[silin284]

I used the home made 100 mM ZnSO4 one.

However, I dont know why solexa recon cutting the 200 bp band. the RNA fragments are mainly 60-100 nt, the 2 adapters are around 60 in total. So the majority of the library should be smaller than 200 bp.

I checked that by PCRing out the non-size selected ligation product and they indeed produce a smear but the strongest band is between 150-200.

[xile]

HI:
I always thought that fragmentation buffer used by Illumina is identical to fragmentation buffer used for cRNA fragmentation step for Affy chips, and stop buffer is just EDTA to chelate cations out.

http://sysbio.harvard.edu/csb/resour...n_Protocol.doc

I agree with you.

[das]

You may be right.
Fragmentation step is 35 minutes during affy procedures and only 5-9 minutes for illumina, so perhaps it is Zn instead of K and Mg that shortens the incubation time.

[Nik]

Yes, stop buffer is 0.5M EDTA but the frag buffer I'm looking and 94c for 8 mins seems excessive. I'm going to try either or both 100mM ZnCl2 in 100mM Tris-HCl pH7 or the Amdion kit using 70c for 15mins. If it is the Affy one I can get loads of that.

[das]

HI:
I always thought that fragmentation buffer used by Illumina is identical to fragmentation buffer used for cRNA fragmentation step for Affy chips, and stop buffer is just EDTA to chelate cations out.

http://sysbio.harvard.edu/csb/resources/downloads/Bauer_Core_GeneChip_Fragmentation_Protocol.doc

[xile]

Hi, we found that fragmentation isn't needed if you just need the count of each mRNA.