I am planning to design primer for His tag. The general format of the forward primer : CGC: Restriction site (GGTACC) :Spacer(GGAGGTACTGA): Start codon (ATG): Six Histidine (CACCATCACCATCACCAT): followed by my gene sequence (CCT GCA GCA ACC ACT TAT):
The problem is that the Tm is getting too high (75 ̊C) for the forward primer
What should I do to adjust the Tm between the forward and the reverse primer?
1. Should I remove the spacer sequence?
2. Should I remove few more base pairs from the gene sequence?
Please let me know. Thank you.
First of all, if you want to calculate Tm you only have to enter the sequence of the primer that really binds to your gene of interest, so in your case CCT GCA GCA ACC ACT TAT. Then, the Tm for e.g. Phusion polymerase is about 59.4°C, so that is absolutely fine.
If you don't need the spacer sequence, e.g. for correct distance to your ribosomal binding site, I would remove it. On the other hand, I would add additional 3 basepairs in the beginning of your primer. Some restriction enzymes need 6 bp distance to the beginning / end of the strand to cut efficiently. You can look this up, which need this, but adding 6 bp always worked in my hands.
So here is the primer I would design:
CGCCGC - GGTACC - ATG - CACCATCACCATCACCAT - CCT GCA GCA ACC ACT TAT
The overhang is not very large, but sometimes the proof reading polymerases start to digest this. Use a hotstart polymerase to be absolutely sure, that this won't happen. I once had this problem but the overhang was almost 80 bp, so I guess in your case it will be fine.
First of all, if you want to calculate Tm you only have to enter the sequence of the primer that really binds to your gene of interest, so in your case CCT GCA GCA ACC ACT TAT. Then, the Tm for e.g. Phusion polymerase is about 59.4°C, so that is absolutely fine.
If you don't need the spacer sequence, e.g. for correct distance to your ribosomal binding site, I would remove it. On the other hand, I would add additional 3 basepairs in the beginning of your primer. Some restriction enzymes need 6 bp distance to the beginning / end of the strand to cut efficiently. You can look this up, which need this, but adding 6 bp always worked in my hands.
So here is the primer I would design:
CGCCGC - GGTACC - ATG - CACCATCACCATCACCAT - CCT GCA GCA ACC ACT TAT
The overhang is not very large, but sometimes the proof reading polymerases start to digest this. Use a hotstart polymerase to be absolutely sure, that this won't happen. I once had this problem but the overhang was almost 80 bp, so I guess in your case it will be fine.
I was wondering, can we replace C and G of spacer region with A and T, to make it AT rich that would help in lowering the Tm? In case If my assumption is wrong, can anyone help in understanding it better?
Anil, your assumption is wrong. As the spacer region does not bind to your gene of interest, it does not effect Tm. Only the bases that actually bind your gene of interest need to be considered for this calculation. Typically, you use 20 bp that bind and then add an overhang. If you want to lower Tm, you have to shorten this binding region to 19 or 18 bp. But this is rarely necessary in my experience, if you don't work with GC-rich organisms, as you can go up to Tm of 72°C.
Thanks Jonas and Jai,
Special thanks to Jonas for highlighting the point, I skipped.
However, after first reaction all the subsequent reactions would include the overhang in template. I was wondering, whether it would affect the Ta for subsequent reactions...
Please forgive me if I am talking non-sense...
"Please forgive me if I am talking non-sense..."
No you are not, but you seem to be overthinking a bit - this is quite straightforward. ;)
[Prithwi, in Guruji's (Dr. Bachhawat) time your place was renamed to Indian Institute of Chemical Biology. Has it been changed back to Bose Inst?]
"you seem to be overthinking a bit"
Probably yes... I give my neurons a break...
The most you may have to do is to bump up the annealing temp suitably after 1-2 initial cycles, just in case if you encounter any unexpected products, which is unlikely.
Dear Ghosh
Please you can also find some information with Gibson assembly cloning (http://www.nature.com/nmeth/journal/v6/n5/abs/nmeth.1318.html, http://nebuilder.neb.com).
Cordialy
Marius
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