pENTR1A Transformation not working

[CambridgeBiochemist]

Dear all,

I have been looking around this site and it seems many people are getting their problems solved..so I will try my luck.

Currently, I am trying to insert my gene (1800bp) into pENTR1A vector.
Gene to replace the lethal ccd gene in pENTR1A

Steps done:
My gene was PCR amplified from pGEX6p-2 vector
--> Agarose gel run (1800bp band seen)
--> PCR reaction purified using Quiagen kit
--> Gene and vector (pENTR1A) digested with SalI and NotI
(FastDigest Enzymes used), ~1hour incubation, 37C, Insert: 50uL reaction (enzyme used: 5/50=10%), Vector: 20uL reaction (enzyme used: 2/20=10%)
---> Agarose gel run -> insert digestion: ~1800bp band seen (expected); vector digestion: ~ 2200bp band seen (as expected) --> both gel purified using Quiagen kit
--> Ligation (Fermantas T4 DNA Ligase) 20uL reaction, 2/20: 10% Ligase used, 1hour, 22C, 1:3 (vector: insert)
--> Transformation 1: 6uL of Ligated DNA into 100uL dh5alpha cells (standard protocol)
--> Gel Run -> 4000bp ligated band seen (though faint) --> gel Purified
--> Transformation 2: 6uL of Gel purified Ligated DNA into 100uL dh5alpha cells

Transformation results:
Transformation 1: One colony
Transformation 2: No colony (surprising since I have pure ligated product, but little agarose might have inhibited transformation?)

Next Steps:
Transformation 1: One colony -> 5mL Overnight Culture, 40ug/mL KAN, 37C 300rpm -> Miniprep ->
-> Absorbance check: 6ng/uL reading
-> Gel check -> No band found

query: How come no plasmid, if I got the colony?
-Undigested pENTR1A vector into dh5aplha cells should not grow due to lethal ccd gene
-Partly digested pENTR1A cannot go inside the cells and pENTR1A cannot ligat to itself since it was cut with two different enzymes..

So how did I get that colony w/o ligated pENTRA1A vector in it?

Next: I doubted I got low plasmid, so was not able to detect it...I did Maxi Prep..(150mL culture)
--> Whole of Maxi prep final solution loaded onto gel -> no band
For sure I don't have ligated pENTR1A in that colony..

Perhaps, undigested pENTR1A got in and that colony is resistant to lethal ccd gene.
or more likely, that colony of dh5alpa is resitant to KAN so it grew..!!

So at the end of the day, I have not got my ligated pENTR1A vector into dh5alpha cells:

That is not yet all: I did transformation controls
(1) dh5cells spreaded on LB plate (no antibiotic) -> many colonies -> dh5 cells are alive
(2) pENTR1A transformed into dh5alpha spreaded on LB KAN plate -> no colonies (as expected, ccd gene in pENTR1A is lethal)
(3) KAN LB plate w/o cells incubated -> no colonies -> so no background colonies
(4) dh5cells spreaded on LB KAN plate -> no colonies -> Kanamycin is working
So the dh5alpha cells and Kanamycin is working alright. But I must admit, I can't say that transformation is working alright since undigested pENTR1A is lethal to cells and as expected I don't get colonies...now that is due to ccd killing cells or KAN killing dh5cells w/o pENTR1A...it's difficult to tell...

However, I did a transformation a month before with a pGEX6P2 vector into dh5alpha and got colonies, so I feel transformation is working..
Taking that forward...that transformation is working....it's something to do with ligation...

I repeated ligation: this time overnight ligation: 4C, 18hours, 1:3 (V:I), 20uL reaction, 5% ligase -> Did two transformations:
T1: 1uL into 50uL cells -> no colonies
T2: 3uL into 50uL cells -> no colonies

So still ligation trouble exists...any suggestions?...You know I have been really working day and night with exp and controls...so I need an expert advice..Thanks in advance..

[hairongxu88]

i am learning