Do I need to sequence after digestion? - (Apr/22/2013 )

I know I should sequence after ligating a PCR product, but what about something that I've digested from one plasmid and ligated into another plasmid? Do I need to sequence through the restriction sites to make sure that everything is fine, or are the chances of this pretty low?

smndtupidisaftr on Mon Apr 22 20:47:47 2013 said:

You could indeed sequence in the end to make sure you are 100% right.
If its indeed your final product and you need to make sure its ok.. than yes, you better confirm it by sequencig.

There are other ways of confirming your result, but I generally try to sequence. It's fast and relatively inexpensive, and provides confidence that you are working with the correct construct.

If I've done a test digest and it looks fine, what would you say the chances are of any problems?

Things are probably fine, but being certain is worth a good deal, in my experience.

smndtupidisaftr on Mon Apr 22 22:12:45 2013 said:

Like said: a sequence test is just something you do to be 100% sure its ok...
If you need it as a expression vector or something for the next 3-4 years to finish your experiment/PhD or whatever than its worth to sequence it.

There are other ways too, but they are often more time consuming and face it: if you sequence it you get the exact result. And its indeed cheap too (cheaper than those other tests most often)

Well i sequence only after transformation and selectivity on agar and after PCR confirmation. But i do it only, when i am sure that the positive transformation is at high chance. if you are constructing plasmid in many steps - adding few inserts, then you should sequence to make sure that your work is going forward...
I do so just because sequencing is pretty expensive as we have to use the company service and we dont have a contract with any of such companies..;.

bigudukaz on Tue Apr 23 08:05:44 2013 said:

What do you call expensive? Because I dont pay that much for sequencing an insert...
The other methodes are more timeconsuming and thus often more expensive.

pito on Tue Apr 23 18:06:29 2013 said:

Well We pay for one sequence ~20 euros and to be sure - it has to overlap so its 20 euros. I preffer beeing on the safe side and do simple colony PCR to know weather there is a correct size insert and choose "better" looking (from PCR) colonies to grow->purify plasmid. Maybe its not usefull, but i was tough to do so in my lab and the reasoning was the one i just wrote

bigudukaz on Wed Apr 24 08:08:14 2013 said:

20 euros?
Thats a lot...
There are companies that offer plasmid sequencing for 5 dollars.