Protocol Online logo
Top : New Forum Archives (2009-): : Molecular Cloning

Sending a TOPO-construct for sequencing? - (Apr/08/2014 )

I have cloned my insert into a TOPO-vector.

Then I digested with 2 restriction-enzymes that targets sites flanking the insert in the TOPO-vector.

I was expecting to get 2 bands (one band being the vector, and the other smaller band the insert).


But what I got was 1 single big band. So probably the digest didn't work.


Question 1)

Could that be because I used 0.3ul of restriction-enzymes instead of 1ul for each 1ug DNA? My supervisor told me to use 0.3ul to save the expensive reagent.


Question 2)

Despite the results from the electrophoresis, we now want to send it for sequencing. And I have been told different things.

a) I haft to construct primers for each 700bp region of the construct (overlapping ones) and ones I get them, I will send them together with the TOPO-construct for sequencing.

b  )I don't need to design anything. Just send the TOPO-construct to the company that does the sequencing


Which one is it now? People are saying different things here (I am gonna talk with my supervisor, but I want to get some input from here as well)


How much plasmid did you used for digestion (in ng/ug) and what RE (EcoRI?) and what conditions (time, temperature)?


Your plasmid is pCR4-TOPO (3956 bp). What is the size of the insert again?


RE is hardly going to cut only one of tho sites, if they are intact, unsufficient RE amount may cause lesses precentage of cut vs uncut vector. But EcoRI cuts like hell even with less enzyme. And in many cases it's enough you increase the incubation time to get everything cut.



The sequencing depends how much the prize differs in this case and what is your experience with primer design. If you are not sure, it's probably better the company does that, though your boss ma have different oponion, if it's too expensive.

Designing primers is easy, if you are proficient in it.

But if you screw it the cost of getting the compete sequence gets higher at the end.