Hi !
I’m beginner in molecular cloning, and I have to make a blunt ligation (I know it’s rather tricky, but there’s no other choice, it’s a specific vector).
I’ve read a lot of protocols and forums, and here is what I’m planning to do.
My questions are about insert and vector preparation, and particulary about purifications : what are the steps where purification is necessary, and after wich steps can I skip purifications ?

Insert :
- PCR with Herculase (proofreading, no A overhangs)
- Purification with Qiagen PCR purification kit
- Digestion with restriction enzyme Pme I (blunt ends)
- Migration on agarose gel, excision and purification of the insert (kit “Wizard SV Gel and PCR Clean-Up System” from Promega)

Vector :
- Digestion SalI/BamHI
- Migration on agarose gel and purification of the band corresponding of the cut vector (Promega kit)
- Blunting with Klenow polymerase
- Purification (kit PCR purification)
- Dephosphorylation with Antartic phosphatase and heat inactivation of the phosphatase

- Estimation of the relative amount of insert and vector by side-by-side gel migration

- Ligation...

What do you think about this part of protocole ? Is there mistakes or possible improvements ?

Thanks in advance !

Helen

Hi Helen!

It looks fine for me.
But you can skip some purification steps in the vector preparation if you like:

- Digestion SalI/BamHI
- Heat inactivation of SalI (BamHI is not affected - when you want to remove it, you can do a purification, with a PCR purification kit)
- Blunting with Klenow polymerase (Klenow works in other buffers, too)
- Heat inactivation
- Dephosphorylation with Antartic phosphatase and heat inactivation of the phosphatase (Antarctic Phosphatase can also be used in other buffers, but ONLY when supplemented with 10X Antarctic Phosphatase Reaction Buffer to a final concentration of 1X)
- Migration on agarose gel and purification of the band corresponding of the cut vector

Good luck!

Chakchel

Hi Helen!

It looks fine for me.
But you can skip some purification steps in the vector preparation if you like:

- Digestion SalI/BamHI
- Heat inactivation of SalI (BamHI is not affected - when you want to remove it, you can do a purification, with a PCR purification kit)
- Blunting with Klenow polymerase (Klenow works in other buffers, too)
- Heat inactivation
- Dephosphorylation with Antartic phosphatase and heat inactivation of the phosphatase (Antarctic Phosphatase can also be used in other buffers, but ONLY when supplemented with 10X Antarctic Phosphatase Reaction Buffer to a final concentration of 1X)
- Migration on agarose gel and purification of the band corresponding of the cut vector

Good luck!

Chakchel

I would advice caution when considering a blunt end ligation. Blunt end ligation is not the easiest thing to do. Is it at all possible to use a strategy using sticky end overhangs? You can add restriction sites to primers, and thus make inserts via PCR which have desired restriction sites.

I know blunt-end cloning is difficult, but I don't have the choice : I must cut my vector with BamHI and there is a BamHI recognition site in the middle of my insert...
Thanks for reply !
Have a nice day

Or

Use BclI
Since you are making your insert by PCR, you could also add BclI sites (which along with BglII) forms compatible ends with BamHI. BclI sites are dam methylated so can't be used by on DNA grown in dam+ cells, but this is not a problem as the insert is made by PCR

Or

Add Xho restriction sites to the PCR insert.
Partial fill the Xho insert. (2bp)
Cut the vector with BamHI, partial fill the bamHI site. (2bp)
Draw out the sites, you will find that by partial filling (with 2bp), the Xho and BamHI sites have become compatible ends.

Or

SalI and XhoI sites form compatible overhangs. Engineer Xho sites into the primers of your insert, cut them and directly insert into the Sal site of your vector (I don't recommend working with SalI ligations, the enzyme is funny. Sucessful ligation is not a certainty)

The options are vaired and many.

Nice post Helen, it's a very good protocol. My only other suggestion would be to make sure that the vector DNA you are starting off with is good quality and not too old. You can check the quality of your vector by screening it by digest, in say 3 or 4 positions around the vector to confirm that all of it is in tact and not degraded. If the digest is not quite right, you can transform the vector into competent cells, plate out, pick a colony to grow overnight and miniprep and screen that. Vector stock quality is important because much like a builder and his starting materials if you start with poor quality vector then you can only get a poor quality vector preparation.

Good luck,
Rob

Thanks everybody for your precious answers !
I'll follow Almasy's suggestion and make cohesive ends by adding a BglII site...

Good luck