sorry for the late reply ~
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There have been 41 items by wdyeo (Search limited from 22-November 18)
Thanks alot for the advice and i really agree with you
But i just found this, to make 10X stock here!
U think it will work?
To make 10 ml of 10x stock
- In 70 % glycerol / 30 % water, dissolve the following:
- 0.606 g Tris-base
- 2.5 g SDS
- Adjust the pH using pH indicator strips to 6.8
- Add 2 mg of Bromophenol Blue and make sure the powder is completely dissolved
- Adjust the final volume to 10 ml with 70 % glycerol / 30 % water before storing at -20°C.
- Add the appropriate volume of a β-mercaptoethanol 100% stock to your samples just before denaturing them at 95°C.
Is there any recipe for home-made 10x Laemmli buffer?
My sample is just too low concentration but the common stock is 2x,4x,5x and 6x...
And is it possible not to use powder form of SDS? I don't know how to dissolve it and we have 20% SDS in solution.
I am using a 4-12% Bis-Tris NuPage (Invitrogen) ready-made gel , and run in MES buffer to enable me to view the protein size of less than 10k Da.
However, when i transferred the gel to the Immobilon PVDF 0.2um using Trans-Blot SD Semi-Dry (Biorad), at 12V for 45 min for the mini gel , the transferring is still incomplete when i stained my gel with coomasie blue after transferring it.
The transfer buffer which I am using is theglyicine , tris base, SDS and methanol.
I did not use the running buffer as suggested for NuPage transfer buffer.
Is it because of the different transfer buffer which causes the inefficiency of the transferring?
I made a silly big mistake by adding twice more the amount of EDTA for the MES buffer...
Will it has much affect on it?
I do not have MES and other chemicals to add to make the amount equivalent for the extra EDTA
I am interested to know whether you put all the isolated RNA (after extraction from Trizol) to the Qiagen RNeasy Mini, by adding the RLT buffer and the subsequent step for the clean up?
I had extracted RNA from Trizol but my ration 260/230 is too low and I am wondering what shall i do to improve the ratio?
I used RNeasy Mini Qiagen to extract RNA from cell culture.
But if i use 26G needle instead of 20G needle (as written down in the handbook) to homogenize the cells in cell culture, will it affects the quality of RNA or i loss more RNA etc?
Edited to make font readable. Bob
I harvested the tissues (such as pancreas, liver and spleen) in order to detect if there is any GFP+ cells
Is it possible to analyse/sort the tissues samples using FACS to quantify the number of GFP+ cells which might be less than 10 cells!?!
I inject GFP stem cells into a normal mouse and i harvested the tissues (such as pancreas, liver and spleen) in order to detect if the GFP+ cells are successfully transplanted/home into the mouse.
Is it possible for me to do RT-PCR or qPCR to detect the level of GFP+ cells? Which is more sensitive because maybe only 1 or 2 GFP+cells in whole tissues..Or any other technique besides immunohistochemistry?
I have no idea if the GFP+ cells are transplanted successfully into the mouse by the way
The problem i am facing now is, i try to identify GFP staining in my tissues and one of the primary antibody is against the GFP and i am supposed to stain the GFP with secondary in green to enhance the GFP signalling but i made a silly mistake, using red for the GFP antibody .
I am doing the immunohistochemistry on the tissues, in the gel about 150um (cut with vibratome).
I made a mistake in my secondary antibody, instead of using secondary anti-rabbit green + secondary anti-mouse red; i used secondary anti-rabbit red and secondary anti-mouse green . I did a double staining for my primary antibodies, one is against rabbit and the other is mouse.
Is there a way to remove the secondary antibody and restain my tissues slice?