9 replies to this topic

[smndtupidisaftr]

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?

[pito]

smndtupidisaftr, on 22 April 2013 - 12:47 PM, said:

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?

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.

[phage434]

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.

[smndtupidisaftr]

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

[phage434]

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

[pito]

smndtupidisaftr, on 22 April 2013 - 02:12 PM, said:

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

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)

[bigudukaz]

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..;.

[pito]

bigudukaz, on 23 April 2013 - 12:05 AM, said:

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..;.

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.

[bigudukaz]

pito, on 23 April 2013 - 10:06 AM, said:

bigudukaz, on 23 April 2013 - 12:05 AM, said:

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..;.

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.

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

[pito]

bigudukaz, on 24 April 2013 - 12:08 AM, said:

pito, on 23 April 2013 - 10:06 AM, said:

bigudukaz, on 23 April 2013 - 12:05 AM, said:

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..;.

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.

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

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

Edited by pito, 24 April 2013 - 01:04 AM.