i need to clone my PCR fragment into the cloning vector pTZ 57R/T....I am not able to add restriction sites to my primer as i am very new to cloning. Please help me out in detaile how to clone PCR fragment into cloning vector.
Dear Srinah
If you want to clone your PCR product into a plasmid of interest,
1. You need to check the multiple cloning site (MCS) of the vector to identify the unique enzymes that do not cut your target gene (your PCR product). This is based on in silico analysis.
2 Design primers for your target gene and include the RE sites of the identified enzymes at the 5' end of each primer immediately next to your gene specific sequence. It is strongly recommended that you choose two different enzymes sites to avoid plasmid recircularisation (which is very common when you use a single enzyme sites on both forward and reverse primers).
3. An average of 4 nucleotides, known as sitting sequence, are needed just before the RE recognition sequence, for efficient cleavage of your PRC product. Most at times, the choice of those nucleotides is arbitrary.
In a nutshell, each primer should be designed as
5' sitting sequence (about 4 nt)----RE site (mostly 6nt).....target gene sequence (18-22 nt).....3'
Hope this helps
Bashir.
Dear Satish U have to add desired restriction site sequences in your forward and reverse primer sequences.
Dear Srinath,
1. when you design your primer pair, you'd better keep Tm of the two primers are the same or very close.
2. you add your desired enzyme site at 5' end of your primer sequences (Note: when you calculate your Tm, you just count your primer sequence complementary to your gene). If your want to directly digest your PCR product for cloning, you'd better add three more bases (any but I prefer AAA) before your enzyme site. (this is for the enzyme digestion purpose)
Good luck!
Dear Srinah
If you want to clone your PCR product into a plasmid of interest,
1. You need to check the multiple cloning site (MCS) of the vector to identify the unique enzymes that do not cut your target gene (your PCR product). This is based on in silico analysis.
2 Design primers for your target gene and include the RE sites of the identified enzymes at the 5' end of each primer immediately next to your gene specific sequence. It is strongly recommended that you choose two different enzymes sites to avoid plasmid recircularisation (which is very common when you use a single enzyme sites on both forward and reverse primers).
3. An average of 4 nucleotides, known as sitting sequence, are needed just before the RE recognition sequence, for efficient cleavage of your PRC product. Most at times, the choice of those nucleotides is arbitrary.
In a nutshell, each primer should be designed as
5' sitting sequence (about 4 nt)----RE site (mostly 6nt).....target gene sequence (18-22 nt).....3'
Hope this helps
Bashir.
Dear All
Thank you very much for your kind response..and it helped me...@Chunhong Chen thank you for the Tm calculation...I was confused and worried about it....@Amit Singh and @Bashir...Thank You Very Much for the detailed explanation you have given..
I have a related question: Can I use a proofreading polymerase to extend such a primer with added RE site, or will the linker containing the restrictionsite be degraded
you should add the sequences of relevant enzyme at the first of primer before ordering to be synthesizes.
Hello Everyone,
I need to know how many nucleotidies should we add before RE recognition sequence when we design primers. Could we add so many nucleotides or there are limitation for adding them. Thanks in advance
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