Re: [ccp4bb] Primer design
A lot of plant genomes are big- wheat for example is 12 Gb, so it may not be quite as trivial as one might expect. cheers, tom Tom Peat Proteins Group Biomedical Program, CSIRO 343 Royal Parade Parkville, VIC, 3052 +613 9662 7304 +614 57 539 419 tom.p...@csiro.au From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> on behalf of Keller, Jacob <kell...@janelia.hhmi.org> Sent: Tuesday, July 25, 2017 1:43 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Primer design >No need of the whole exome. Sequencing The second PCR product will do the job >I guess. Second PCR (from the cDNA pool) with specific forward primer and and >oligodA reverse primer. Surely a matter of less than $3 $3 is a major understimation, but I see your point. On the other hand, it is important to consider new technologies, and it would be a service to the scientific community to publish the exome somewhere, so other researchers would not have to spend their $3. JPK Best, DKG - Original Message - From: "Jacob Keller" <kell...@janelia.hhmi.org> To: "Debasish Kumar Ghosh" <dkgh...@cdfd.org.in>, CCP4BB@JISCMAIL.AC.UK Sent: Monday, July 24, 2017 6:48:25 PM Subject: RE: Primer design Or sequence the whole exome for what, $500-1000? JPK -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Debasish Kumar Ghosh Sent: Monday, July 24, 2017 7:43 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Primer design Dear Syed, The process is very trivial to clone your gene of interest. Assuming your gene is transcribed as mono-cistronic mRNA, take the oligodT primer as reverse primer. First isolate the total RNA from the tissue or cells and do the cDNA synthesis using oligodT primer followed by gene specific PCR with forward primer and oligodA primer from the cDNA pool. Sequence the PCR product to get to know the first stop codon in the ORF. Making the specific reverse primer from the sequence is then just matter of time. Hope this helps. Best wishes, Debasish CSIR- Senior Research Fellow (PhD Scholar) C/o: Dr. Akash Ranjan Computational and Functional Genomics Group Centre for DNA Fingerprinting and Diagnostics Hyderabad, INDIA Email(s): dkgh...@cdfd.org.in, dgho...@gmail.com Telephone: 0091-9088334375 (M), 0091-40-24749396 (Lab) Lab URL: http://www.cdfd.org.in/labpages/computational_functional_genomics.html - Original Message - From: "syed ibrahim" <048c02cac012-dmarc-requ...@jiscmail.ac.uk> To: CCP4BB@JISCMAIL.AC.UK Sent: Monday, July 24, 2017 4:56:00 PM Subject: [ccp4bb] Primer design Hello All I am interested in cloning a gene from a plant. I searched the database only partial sequence is available, ie: for 160 residues only. The full length of the protein is around 570 residues. I designed forward primer and I have no clue to design reverse primer. Any help Thank you Syed
Re: [ccp4bb] Primer design
>No need of the whole exome. Sequencing The second PCR product will do the job >I guess. Second PCR (from the cDNA pool) with specific forward primer and and >oligodA reverse primer. Surely a matter of less than $3 $3 is a major understimation, but I see your point. On the other hand, it is important to consider new technologies, and it would be a service to the scientific community to publish the exome somewhere, so other researchers would not have to spend their $3. JPK Best, DKG - Original Message - From: "Jacob Keller" <kell...@janelia.hhmi.org> To: "Debasish Kumar Ghosh" <dkgh...@cdfd.org.in>, CCP4BB@JISCMAIL.AC.UK Sent: Monday, July 24, 2017 6:48:25 PM Subject: RE: Primer design Or sequence the whole exome for what, $500-1000? JPK -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Debasish Kumar Ghosh Sent: Monday, July 24, 2017 7:43 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Primer design Dear Syed, The process is very trivial to clone your gene of interest. Assuming your gene is transcribed as mono-cistronic mRNA, take the oligodT primer as reverse primer. First isolate the total RNA from the tissue or cells and do the cDNA synthesis using oligodT primer followed by gene specific PCR with forward primer and oligodA primer from the cDNA pool. Sequence the PCR product to get to know the first stop codon in the ORF. Making the specific reverse primer from the sequence is then just matter of time. Hope this helps. Best wishes, Debasish CSIR- Senior Research Fellow (PhD Scholar) C/o: Dr. Akash Ranjan Computational and Functional Genomics Group Centre for DNA Fingerprinting and Diagnostics Hyderabad, INDIA Email(s): dkgh...@cdfd.org.in, dgho...@gmail.com Telephone: 0091-9088334375 (M), 0091-40-24749396 (Lab) Lab URL: http://www.cdfd.org.in/labpages/computational_functional_genomics.html - Original Message - From: "syed ibrahim" <048c02cac012-dmarc-requ...@jiscmail.ac.uk> To: CCP4BB@JISCMAIL.AC.UK Sent: Monday, July 24, 2017 4:56:00 PM Subject: [ccp4bb] Primer design Hello All I am interested in cloning a gene from a plant. I searched the database only partial sequence is available, ie: for 160 residues only. The full length of the protein is around 570 residues. I designed forward primer and I have no clue to design reverse primer. Any help Thank you Syed
Re: [ccp4bb] Primer design
No need of the whole exome. Sequencing The second PCR product will do the job I guess. Second PCR (from the cDNA pool) with specific forward primer and and oligodA reverse primer. Surely a matter of less than $3 Best, DKG - Original Message - From: "Jacob Keller" <kell...@janelia.hhmi.org> To: "Debasish Kumar Ghosh" <dkgh...@cdfd.org.in>, CCP4BB@JISCMAIL.AC.UK Sent: Monday, July 24, 2017 6:48:25 PM Subject: RE: Primer design Or sequence the whole exome for what, $500-1000? JPK -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Debasish Kumar Ghosh Sent: Monday, July 24, 2017 7:43 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Primer design Dear Syed, The process is very trivial to clone your gene of interest. Assuming your gene is transcribed as mono-cistronic mRNA, take the oligodT primer as reverse primer. First isolate the total RNA from the tissue or cells and do the cDNA synthesis using oligodT primer followed by gene specific PCR with forward primer and oligodA primer from the cDNA pool. Sequence the PCR product to get to know the first stop codon in the ORF. Making the specific reverse primer from the sequence is then just matter of time. Hope this helps. Best wishes, Debasish CSIR- Senior Research Fellow (PhD Scholar) C/o: Dr. Akash Ranjan Computational and Functional Genomics Group Centre for DNA Fingerprinting and Diagnostics Hyderabad, INDIA Email(s): dkgh...@cdfd.org.in, dgho...@gmail.com Telephone: 0091-9088334375 (M), 0091-40-24749396 (Lab) Lab URL: http://www.cdfd.org.in/labpages/computational_functional_genomics.html - Original Message - From: "syed ibrahim" <048c02cac012-dmarc-requ...@jiscmail.ac.uk> To: CCP4BB@JISCMAIL.AC.UK Sent: Monday, July 24, 2017 4:56:00 PM Subject: [ccp4bb] Primer design Hello All I am interested in cloning a gene from a plant. I searched the database only partial sequence is available, ie: for 160 residues only. The full length of the protein is around 570 residues. I designed forward primer and I have no clue to design reverse primer. Any help Thank you Syed
Re: [ccp4bb] Primer design
Or sequence the whole exome for what, $500-1000? JPK -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Debasish Kumar Ghosh Sent: Monday, July 24, 2017 7:43 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Primer design Dear Syed, The process is very trivial to clone your gene of interest. Assuming your gene is transcribed as mono-cistronic mRNA, take the oligodT primer as reverse primer. First isolate the total RNA from the tissue or cells and do the cDNA synthesis using oligodT primer followed by gene specific PCR with forward primer and oligodT primer from the cDNA pool. Sequence the PCR product to get to know the first stop codon in the ORF. Making the specific reverse primer from the sequence is then just matter of time. Hope this helps. Best wishes, Debasish CSIR- Senior Research Fellow (PhD Scholar) C/o: Dr. Akash Ranjan Computational and Functional Genomics Group Centre for DNA Fingerprinting and Diagnostics Hyderabad, INDIA Email(s): dkgh...@cdfd.org.in, dgho...@gmail.com Telephone: 0091-9088334375 (M), 0091-40-24749396 (Lab) Lab URL: http://www.cdfd.org.in/labpages/computational_functional_genomics.html - Original Message - From: "syed ibrahim" <048c02cac012-dmarc-requ...@jiscmail.ac.uk> To: CCP4BB@JISCMAIL.AC.UK Sent: Monday, July 24, 2017 4:56:00 PM Subject: [ccp4bb] Primer design Hello All I am interested in cloning a gene from a plant. I searched the database only partial sequence is available, ie: for 160 residues only. The full length of the protein is around 570 residues. I designed forward primer and I have no clue to design reverse primer. Any help Thank you Syed
Re: [ccp4bb] Primer design
Dear Syed, The process is very trivial to clone your gene of interest. Assuming your gene is transcribed as mono-cistronic mRNA, take the oligodT primer as reverse primer. First isolate the total RNA from the tissue or cells and do the cDNA synthesis using oligodT primer followed by gene specific PCR with forward primer and oligodT primer from the cDNA pool. Sequence the PCR product to get to know the first stop codon in the ORF. Making the specific reverse primer from the sequence is then just matter of time. Hope this helps. Best wishes, Debasish CSIR- Senior Research Fellow (PhD Scholar) C/o: Dr. Akash Ranjan Computational and Functional Genomics Group Centre for DNA Fingerprinting and Diagnostics Hyderabad, INDIA Email(s): dkgh...@cdfd.org.in, dgho...@gmail.com Telephone: 0091-9088334375 (M), 0091-40-24749396 (Lab) Lab URL: http://www.cdfd.org.in/labpages/computational_functional_genomics.html - Original Message - From: "syed ibrahim" <048c02cac012-dmarc-requ...@jiscmail.ac.uk> To: CCP4BB@JISCMAIL.AC.UK Sent: Monday, July 24, 2017 4:56:00 PM Subject: [ccp4bb] Primer design Hello All I am interested in cloning a gene from a plant. I searched the database only partial sequence is available, ie: for 160 residues only. The full length of the protein is around 570 residues. I designed forward primer and I have no clue to design reverse primer. Any help Thank you Syed
Re: [ccp4bb] Primer design
Have to do primer walking in a cdna library first. Artem On Jul 24, 2017 7:26 AM, "syed ibrahim" < 048c02cac012-dmarc-requ...@jiscmail.ac.uk> wrote: Hello All I am interested in cloning a gene from a plant. I searched the database only partial sequence is available, ie: for 160 residues only. The full length of the protein is around 570 residues. I designed forward primer and I have no clue to design reverse primer. Any help Thank you Syed
[ccp4bb] Primer design
Hello All I am interested in cloning a gene from a plant. I searched the database only partial sequence is available, ie: for 160 residues only. The full length of the protein is around 570 residues. I designed forward primer and I have no clue to design reverse primer. Any help Thank you Syed
