6 replies to this topic

[Nghia]

Hi All,

I am trying to sub-clone my insert  with Hind3 and Nde I into the  MCS site of pBluescript.  But unfornaturely
there's no Nde I  site in the MCS of SK.  How do I go about it! or should I try onther vector with H3 and Nde I sites.

Thanks a lot for any advide or help.

Dan

[perneseblue]

if you have another vector with the required site, it would be faster to use that.

However if you can't find a vector, than the standard thing to do would be to PCR amplify your insert with primers carrying the desired restriction sites on the 5'end. Remember to add a guard sequence, as restriction sites need to be skirted by a few bp to enable the site to be cut efficiently. A rule of the thumb is to use 6bp. Many commonly used restriction sites can work with less, but a few such as NotI and NdeI required 8bp for efficient digest. See NEB technical guide.

eg primer

5' -6bp guard seq -  restriction site - template binding sequence -3'

[NemomeN]

Another way is to fill in your insert to make it blunt and then clone into the EcoRV site and check for orientation.

[Nghia]

Hi perneseblue,

Thank your very much for your advise!  But I don't know about the insert's sequence how could I design the primers to do PCR?  I found out 1 vector with H3 site in the MCS and another  Nde I site far away (~ 400bp from the MCS including T7).  Theorecially I can subclone H3/Nde I insert into the Vector,  but I think I can not do sequencing because T7 would be gone. or should I design other primers to do sequencing the insert?

Ideally I would like to subclone into SK.  Could you please provide me some detail about that.

I appreciate your help and hints.

Have  a wonderful weekend.

Dan  

perneseblue, on Feb 27 2009, 03:29 PM, said:

if you have another vector with the required site, it would be faster to use that.

However if you can't find a vector, than the standard thing to do would be to PCR amplify your insert with primers carrying the desired restriction sites on the 5'end. Remember to add a guard sequence, as restriction sites need to be skirted by a few bp to enable the site to be cut efficiently. A rule of the thumb is to use 6bp. Many commonly used restriction sites can work with less, but a few such as NotI and NdeI required 8bp for efficient digest. See NEB technical guide.

eg primer

5' -6bp guard seq -  restriction site - template binding sequence -3'

[phage434]

You do know the sequence of your vector, so it is easy to PCR the vector backbone and use the primers to insert whatever restriction sites you'd like in the cloning site.  Make sure to check there are not other sites for those enzymes in the vector backbone.

[T C]

Hey

I use pNEB193 or a construct wherein we inserted the MCS of PET (containing both Nhe I and Nde I) in pBS.

Best
TC

Nghia, on Feb 28 2009, 04:27 AM, said:

Hi All,

I am trying to sub-clone my insert  with Hind3 and Nde I into the  MCS site of pBluescript.  But unfornaturely
there's no Nde I  site in the MCS of SK.  How do I go about it! or should I try onther vector with H3 and Nde I sites.

Thanks a lot for any advide or help.

Dan

[scolix]

Try to clone the vector virtually using some program like Vector NTI and it should give you a better idea of the sites. Even if you switch to a different vector check the sites again.