What is the relationship between the average size of the polyethylene glycol molecules and the size of the plasmid on the transformation efficiency of protoplasts?
I'm working on PEG 6000 and the size of the plasmid is around 8kbp. However, I did not transfect successfully. My subject is Penicillium marneffei.
Seems like there is not much effect
for smaller size can go inside the cell
however too big size can increase viscosity and water pressure and and availability of Oxygen
Also in a paper of Chung et al. (1989) they compare different PEGs and find PEG3350 and PEG8000 as best working for E.coli
see links-
http://www.protocol-online.org/biology-forums/posts/26634.html
https://biology.stackexchange.com/questions/1995/why-is-peg-important-for-efficient-yeast-transformation
Hello! I want to quantify by ELISA the secreted (from platelets poor plasma) and the non-secreted (from platelet lysate) PF4 before and after TRAP stimulation. I will use the ELISA from R&D...
03 March 2021 1,499 2 View
Hi, could anyone recommend a plasmid and/or protocol for reporter gene assay in S. cerevisiae? I want to assess the effect of growth conditions on a transcription factor, so I want to clone it`s...
01 March 2021 210 1 View
I have a set of stably transfected cell lines all transfected with plasmids containing GFP tags on the C terminus. During a western blot using anti-GFP antibody, one of my plasmids has dissociated...
01 March 2021 9,310 4 View
I am going to have a expression cloning of mammalian gene by using shuttle plasmid to transforming the E.coli However I don't know I should only inserting the Coding sequence ,or I can...
28 February 2021 5,440 3 View
I transfected my LNCaP-WT cells with 3 shRNA plus their NTC two weeks ago and split two puromycin selected cell plates on Friday last week(Feb 26). I checked for GFP in the cells, and they all...
28 February 2021 4,949 3 View
Hello, Is it possible to use pUC19 as a transfer vector to be packed in using the second generation viral particles packaging system( pMD2.G; psPAX2 plasmids)? As far as I understand it there is...
28 February 2021 4,868 2 View
28 February 2021 2,534 2 View
When using a lentiviral vector for inducible expression of genes using the Tet-On system. In the literature, I saw a widely used of pLVX-TetOne-Puro Plasmid, is there a reason why people prefer to...
25 February 2021 1,011 3 View
After transfection with the plasmid ( linearized ) and subcloning of the cell lines, RNA was extracted from the cell and then reverse transcripted to cDNA. When PCR reactions were run to verify...
25 February 2021 5,712 3 View
I have a large 16 kb plasmid, which I need to transfect into PC12 cells. Lipofectamin 2000 didn't work and with GFP alone the transfection rate is very low. I also tried the neon invitrogen...
25 February 2021 6,635 5 View