Ok I proceeded with my cloning work.
After checking for my insert using EcoRI digestion, my next step to do was to digest my TOPO+insert recombinant with NdeI and XhoI enzymes to free my insert with 5' overhangs as result of the double digestion using the NdeI and XhoI enzymes.
My protocol for this part is as follows:
3ug of DNA (concentration being around 150-160 ng/uL)
3uL 10X buffer H (both enzymes compatible with this buffer)
top up reaction volume to 30uL
Incubated digestion at 37 in a thermocycler for 4 hours.
My vector which I will subclone my insert into was also double digested with NdeI and XhoI.
I performed a sequential digestion where NdeI was added first, checked on gel after 1.5 hours, then XhoI was added.
I also single digested the same vector with NdeI only for later purposes to be used as a control in my ligation reactions.
Total incubation time was 4 hours as well.
The protocol for my sequential digestion of pET vector is as follows:
3ug of pET 14b vector (0.5ug/uL)
3uL 10X buffer H
top up to 30uL
The single digestion is exact as above protocol, but no XhoI was added.
For the pET 14b vector digestion, a dephosphorylation reaction was also done. I used a 0.2U/uL calf intestial alkaline phosphatase, and I added 0.5-1uL of the enzyme to my digestions before running it on a gel. I incubated at 37C for 5 min. Then to inactivate the phosphatase I added EDTA to an equal final concentration of MgCl2 (found in buffer H) and incubated at 65C for 15 min.
For all the above work, after running on a gel I seem to get clear bands and seem to be at the right size. But after gel extraction and purification I obtained really low concentrations and very very faint bands (not seen on gel image especially for my insert). My concentrations for my insert were as low as 5.8 and 6.8ng/uL. My vector concentrations were 15.7ng/uL for the single enzyme digested one and 23.3ng/uL for the double digested one. I am worried to proceed to ligation with such low concentrations what should I do? By the way my gel extraction purification is a kit from Promega called Wizard SV Gel and PCR Clean-Up System
To refer to gel images
In order from first attached to last
For the sequential and single enzyme digestion of pET 14b with NdeI
The gel is from left to right: 1kb ladder, NdeI single digested pET 14b, NdeI sequentially digested pET 14b, undigested pET 14b.
For the TOPO+my insert NdeI and XhoI double digestion, pET 14b single digestion, and pET 14b double digestion NdeI and XhoI
The gel is from left to right: 1kb ladder, TOPO+my insert dble digestion, TOPO+my insert dble digestion, single digested pET 14b, dble digested pET 14b
And the last image is same as the above after gel extraction and clean-up
The gel is from left to right: same as above. As you can see the first two bands are not visible (they were a bit visible as I played with contrast of image, but almost not to be seen)
Thanks Drekketh for your help again.