6 replies to this topic

[wntiong]

Hi, i need to get 10ul of total rna for my experiment, but very often i tried the speedvac drying method, the rna will loss from 30 to 50%. Since my samples are precious, do you think i can add glycogen into my sample and speedvac together? If can, how much glycogen i should add? Will the glycogen affect the microarray experiment or rna degradation?

Kindly advice. Thanks

[gogreen]

wntiong, on Nov 3 2009, 02:32 PM, said:

Hi, i need to get 10ul of total rna for my experiment, but very often i tried the speedvac drying method, the rna will loss from 30 to 50%. Since my samples are precious, do you think i can add glycogen into my sample and speedvac together? If can, how much glycogen i should add? Will the glycogen affect the microarray experiment or rna degradation?

Kindly advice. Thanks

Hi wntiong,

if you want to reduce the volume to the required amount, using a speedvac is the best way to go..I don't think you would lose any RNA in that process...But make sure you don't dry it too much and then try to resuspend it, it will be hard to redissolve the RNA once its quite dried up.

Glycogen is a co-precipitant which can be used during precipitations when u have low amounts of DNA or RNA so that you can visualize a pellet of glycogen and its assumed that RNA/DNA will be precipitated at the same place and you alleviate the risk of pippeting that out! I believe using glycogen in a speedvac is not going to help. Glycogen will not affect microarray experiments or cause/avoid RNA degradation.

[wntiong]

gogreen, on Nov 3 2009, 06:44 PM, said:

wntiong, on Nov 3 2009, 02:32 PM, said:

Hi, i need to get 10ul of total rna for my experiment, but very often i tried the speedvac drying method, the rna will loss from 30 to 50%. Since my samples are precious, do you think i can add glycogen into my sample and speedvac together? If can, how much glycogen i should add? Will the glycogen affect the microarray experiment or rna degradation?

Kindly advice. Thanks

Hi wntiong,

if you want to reduce the volume to the required amount, using a speedvac is the best way to go..I don't think you would lose any RNA in that process...But make sure you don't dry it too much and then try to resuspend it, it will be hard to redissolve the RNA once its quite dried up.

Glycogen is a co-precipitant which can be used during precipitations when u have low amounts of DNA or RNA so that you can visualize a pellet of glycogen and its assumed that RNA/DNA will be precipitated at the same place and you alleviate the risk of pippeting that out! I believe using glycogen in a speedvac is not going to help. Glycogen will not affect microarray experiments or cause/avoid RNA degradation.

Thanks,

But the problem is i did lost some of my rna during speedvac without glycogen. how long i should set to make sure no rna is losing? is it because i speedvac too long?

[mdfenko]

wntiong, on Nov 3 2009, 07:07 PM, said:

But the problem is i did lost some of my rna during speedvac without glycogen. how long i should set to make sure no rna is losing? is it because i speedvac too long?

did you flush the side of the tube with the remaining sample? the rna may have stuck to the sides of the tube.

Edited by mdfenko, 05 November 2009 - 09:10 AM.

[gogreen]

wntiong, on Nov 4 2009, 06:37 AM, said:

gogreen, on Nov 3 2009, 06:44 PM, said:

wntiong, on Nov 3 2009, 02:32 PM, said:

Hi, i need to get 10ul of total rna for my experiment, but very often i tried the speedvac drying method, the rna will loss from 30 to 50%. Since my samples are precious, do you think i can add glycogen into my sample and speedvac together? If can, how much glycogen i should add? Will the glycogen affect the microarray experiment or rna degradation?

Kindly advice. Thanks

Hi wntiong,

if you want to reduce the volume to the required amount, using a speedvac is the best way to go..I don't think you would lose any RNA in that process...But make sure you don't dry it too much and then try to resuspend it, it will be hard to redissolve the RNA once its quite dried up.

Glycogen is a co-precipitant which can be used during precipitations when u have low amounts of DNA or RNA so that you can visualize a pellet of glycogen and its assumed that RNA/DNA will be precipitated at the same place and you alleviate the risk of pippeting that out! I believe using glycogen in a speedvac is not going to help. Glycogen will not affect microarray experiments or cause/avoid RNA degradation.

Thanks,

But the problem is i did lost some of my rna during speedvac without glycogen. how long i should set to make sure no rna is losing? is it because i speedvac too long?

How do u say that u lost rna by speedvac without glycogen?? did u have a with glycogen control??? I guess glycogen isnt going to help you during a vacuum concentration..As told by above post,you should try mixing the liquid onto the walls of the tubes and pipette for sometime

[HomeBrew]

Not sure how you could lose RNA by the speedvac -- RNA does not evaporate.  How did you conclude that RNA was lost?

[Helios]

i agree to the fact that that speed vac doesnt evaporate rna......as for glycogen precipitation of rna, i normally use 5ug and it works well....plus glycogen doesnt interfere with either od260/280 od or with the microarray experiment....