finding the suitable RE - first timer (Apr/22/2009 )

Hi,

I am going to find the suitable RE for the sequence that I am going to cut and put it into pBluescript II KS(+), but I am not sure how to read the MCS and also how am i going to pick the best RE for my sequence. Is anyone over here can guide and help me? Thanks.

Thanks in advance. Any suggestion and guidance really very much appreciated.

Is your sequence from a PCR product or from another plasmid. If you have the sequence you can use any tool to run a restriction analysis such webcutter from NEB. You need to known the MCS on pBluescript II Ks to see which sites are compatable. Restriction enzymes for cloning are usually 6-cutter and only cut only in the sequence.

The sequence will be from cDNA. Oh, this means, i have to check the mcs providing which re to cut, then i choose from the mcs re to screen the suitable re out? I am using some programme called sequencher. I found the NEB cutter website too. I will try it, and will see how it goes. Thanks.


Sp

cheerioet on Apr 22 2009, 01:29 AM said:

Sequencher is ok. Pick the REs that cut as "outside " as possible of the cDNA. then u will get the most out of the cDNA.

hanming86 on Apr 22 2009, 07:36 PM said:

Cut as "outside" means? cut the most far ends for the sequence? I thought it is also advisable to pick up cutter that is a frequent cutter and the size not too small? Can give me some example? Thanks in advance.

Cheerioet

yea that could be done too. do u know anything about the cDNA.

i do wonder if u could attemp a TA cloning on the amplified cDNA. that would save a lot lot of time .

hanming86 on Apr 23 2009, 12:39 AM said:

the cDNA is a rt-pcr product of FRNA by using commercial kit such as monsterscript. I am not so sure if TA cloning is applicable but i can try to ask my supervisor about it, since he suggested to use pbluescript II KS (+)initially.

Thanks...