6 replies to this topic

[cheerioet]

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.

[pcrman]

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.

[cheerioet]

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

[hanming86]

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

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

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.

[cheerioet]

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

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

[hanming86]

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 .

[cheerioet]

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

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 .

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...