Doing 5' and 3' RACE with kit is bit expensive .
can anyone share protocol of 5' and 3' RACE PCR without Kit or some literature regarding.
I am trying to amplify unknown 3' and 5' region of my gene but not successful. I tried several times but i didnt amplify even 3' unknown region also.
please guide me.
Thanks and regards
Harshraj
Hello Harshraj,
You can make your own 5' RACE and 3' RACE kit based on following paper attached.
3' RACE is much easier you can do it much easily just by adding anchor after oligo dT and using complementarity bases of anchor as one primer and your Gene-specific primer. Use of step out PCR can reduce non-specific bands.
5' RACE can be easily done according to the following paper. which uses a template switching methodology almost similar to Clonetech Smarter RACE.
But however you have to get some particular enzymes and some special probes/ primers (which may need HPLC purification). You need to be careful in designing probes or template switching primer or anchor oligo dT that, those nucleotide sequences should not present in the genome of particular organisms you are working with.
1. M- Mul reverse transcriptase with terminal transferase activity ex: Superscript II or revertid M- Mul RT H minus
2. Get High Fidelity polymerase (I used Phusion Hot Start II High Fidelity DNA Polymerase, you can any similar product) advantage of this enzyme you can amplify RACE product even higher than 1 kb easily and has very short amplification time and buffer and enzymes used automatically makes annealing temperature of primer very high there by increasing the specificity of primers.
I can help you more in this
Let me know, from which organisms RNA you want to perform RACE.
There are many research papers available on RACE protocols. A paper attached here is based on clontech 5' RACE protocol is really good and it is easy to perform.
I have done this using a specific primer that anchors within a region of characterized sequence facing towards the uncharacterized region. I used only this primer and tried a range of different annealing temperatures. At an appropriate annealing temperature the specific primer will miss-prime somewhere upstream (or downstream for 3' RACE) in the region of non-characterized DNA. Its useful to have 3 or 4 different primers with differing 3' regions to increase the chances of a miss prime close enough to produce a PCR product. If the annealing temperature is too low, you will get multiple bands so you need to go as high as you can go and still get a PCR product.
Hello Harshraj,
You can make your own 5' RACE and 3' RACE kit based on following paper attached.
3' RACE is much easier you can do it much easily just by adding anchor after oligo dT and using complementarity bases of anchor as one primer and your Gene-specific primer. Use of step out PCR can reduce non-specific bands.
5' RACE can be easily done according to the following paper. which uses a template switching methodology almost similar to Clonetech Smarter RACE.
But however you have to get some particular enzymes and some special probes/ primers (which may need HPLC purification). You need to be careful in designing probes or template switching primer or anchor oligo dT that, those nucleotide sequences should not present in the genome of particular organisms you are working with.
1. M- Mul reverse transcriptase with terminal transferase activity ex: Superscript II or revertid M- Mul RT H minus
2. Get High Fidelity polymerase (I used Phusion Hot Start II High Fidelity DNA Polymerase, you can any similar product) advantage of this enzyme you can amplify RACE product even higher than 1 kb easily and has very short amplification time and buffer and enzymes used automatically makes annealing temperature of primer very high there by increasing the specificity of primers.
I can help you more in this
Let me know, from which organisms RNA you want to perform RACE.
There are many research papers available on RACE protocols. A paper attached here is based on clontech 5' RACE protocol is really good and it is easy to perform.
Thank you Richard and Rajesh
@ Rajesh- I am working on salt tolerant plant Puccinellia tenuiflora.
I read Pinto et al article. its interesting. thank you
can you please guide me more to design TSO primer as genome sequence of Puccinellia tenuiflora is not available.
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