How to add restriction sites to primers?

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

In most cases itis only 4 nucleotides

I also want to ask the same question with Hamza Elati, thank you

You advised using the Snapgene software trial version. It is easy and fast to design the primers and add restriction enzymes.

https://www.snapgene.com

Check out this link. Provides a detailed explanation with the protocol

Hi Srinath Mote ,

pTZ 57R/T is a TA cloning vector. you can simply use final extension hold during PCR to add A overhangs (Taq pol adds A overhangs) and use that amplicon for cloning.

Hope this helps.

Muhammad Bashir Bello Can you please let me if the 5' sitting sequence can be AAAA or TTTT?

Sarmishta Majumdar , as much as possible runs such as AAAA or TTTT should be avoided

Muhammad Bashir Bello Okay.. then what do you suggest to keep as the 5' sitting sequence? I am trying to design primers for certain genes and have to introduce certain RE sites specific to my vector

If you can please let me know

Muhammad Bashir Bello Also could you let me know the reason for avoiding AAAA or TTTT runs?

Interesting ! but what if the insert ( gene of interest) is too long 1939 bp, can we still design a primer with that?

I have to subclone a gene of 1939bp from one vector to another.

Thank you for your clarification.

Sarmishta Majumdar can you be more specific?what is the size of your gene? what vector do you want to use? etc

Kevin Tindo Koffi 1939bp is not too long. what is the destination vector? that will help in guiding you on your cloning strategy

Muhammad Bashir Bello the size of my genes is 0.7KB and 1.5KB, I'm trying to develop my own vector and that is a mammalian expression vector which is bicistronic in nature. I am trying to clone my gene of interest into the two different MCS sites in my vector. So accordingly, I have to add the RE sites to these genes using PCR. I have already designed primers (insilico) which is 5'-- REsite-- target sequence--3'. I need you guidance on the sitting sequence that needs to be added upstream to RE site in the primers.

Sarmishta Majumdar , the sitting sequences can be any random nucleotides of about 4-6 nucleotides (eg TGGA), so long as they do not recreate another RE site that may interfare with your subcloning. Also make sure you use different RE sites for the two MCS since your vector is bicistronic. Once you do that, I think you are good to go. Best of luck

Muhammad Bashir Bello yea, I have ensured that the 2MCS have different RE sites. I was just a little confused about the sitting sequence. I have observed people doing TA cloning with the amplicon produced. Is that required or I can directly amplify using a primer with (5'-sitting sequence--RE site-3') and digest it for ligation?

Yes you can directly just PCR amplify using the designed primers and then proceed with the cloning. However make sure you use high fidelity DNA polymerases for the PCR not Taq polymerse. Probably those that use TA cloning used Taq polymerase in their PCR. Goodluck Sarmishta Majumdar

Muhammad Bashir Bello Thanks a lot!

Muhammad Bashir Bello Hi, can I know what difference will it make if use the Taq polymerase and the high fidelity DNA polymerase. Does it produce more accurate and high yield of our target insert?