gateway cloning - (Jul/28/2009 )

Hi everybody please help me
I am facing few problems with gateway cloning I have been struggling with this for past a month i tried different combinations - I cloned in my 1.1 kb insert into the entry clone(kan resistance),performed LR cloning to insert it into destination clone (binary vector)- and i got plenty of colonies only one fourth of them grew and when i miniprep DNA concentration is low and when I check for the presence of insert I do not see my insert band
Kindly help me I will be grateful to you all for every suggestion u provide
Thanks in advance

collen

run the LR reaction overnight to get more positive insertion events and therefore transformants

What is the destination vector resistance?

also with minipreps make sure your culture is not too old (o/night should be okay) and don't try and extract from too much culture, I use 1.5ml as a standard and they are fine

Arqwen on Aug 6 2009, 02:14 AM said:

Thank you very much I wil make sure the cultures are not old the resistance for my destination vector is spectinomycin

collen on Aug 6 2009, 12:20 PM said:

Thank you Argwen I have another question to ask- my destination vector is 11.2kb and entry vector is 2.7kb and I have been following protocol from invitrogen
Entry clone (50-150 ng) 1-7 μl
Destination vector (150 ng/μl) 1 μl
TE buffer, pH 8.0 to 8 μl
2. Thaw on ice the LR Clonase™ II enzyme mix for about 2 minutes. Vortex the LR Clonase™ II enzyme mix briefly twice (2 seconds each time).
3. To each sample (Step 1, above), add 2 μl of LR Clonase™II enzyme mix to the reaction and mix well by vortexing briefly twice. Microcentrifuge briefly.
4. Incubate reactions at 25°C for 1 hour.
5. Add 1 μl of the Proteinase K solution to each sample to terminate the reaction. Vortex briefly. Incubate samples at 37°C for 10 minutes.

Transformation
I transform them into Top 10 competant cells 1 microlitre of LR cloning reaction to 100 microlitre Top10 competant cells .Following the protocol mentioned below Incubate on ice for 30 minutes. Heat-shock cells by incubating at 42°C for 30 seconds. Add 250 μl of S.O.C. Medium and incubate at 37°C for 1 hour with shaking. Plate 20 μl and 100 μl of each transformation onto selective plates.
I used 150ng of both the entry clone and destination vector but my destination vector is 11.2kb and entry vector 2.7kb so has the large size of destination vector anything to do with reduced transformation efficiency?
Kindly guide me
Thank you very much