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finding the suitable RE


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#1 cheerioet

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Posted 21 April 2009 - 09:29 PM

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.

#2 pcrman

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Posted 21 April 2009 - 09:59 PM

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.

#3 cheerioet

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Posted 22 April 2009 - 01:29 AM

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

#4 hanming86

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Posted 22 April 2009 - 03:36 AM

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.
Lab + Coffee + Music = Bliss

#5 cheerioet

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Posted 22 April 2009 - 04:17 AM

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

#6 hanming86

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Posted 22 April 2009 - 08:39 AM

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 .
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#7 cheerioet

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Posted 22 April 2009 - 05:51 PM

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...




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