long DNA amplification - (Oct/29/2009 )
I have one protein having 2700 aa so corresponding gene have around 9kb length. i have to clone this gene in to E. coli.
can anyone suggest hw can i amplify this much length of DNA ??
thanks in advance for ur suggestions??
-sud373-
sud373 on Oct 29 2009, 04:47 PM said:
I have one protein having 2700 aa so corresponding gene have around 9kb length. i have to clone this gene in to E. coli.
can anyone suggest hw can i amplify this much length of DNA ??
thanks in advance for ur suggestions??
can anyone suggest hw can i amplify this much length of DNA ??
thanks in advance for ur suggestions??
Well by increasing the extension time!!!
and add may be more polemerase than usual so that it does not get exhausted in between!!!
-Pradeep Iyer-
not to forget dNTPs too in excess!!!
-Pradeep Iyer-
If it's a cDNA from a common species (human, mouse), I would recommend looking for a full length cDNA clone in MGC, Origene, ... or any other company. I think the cost will outweigh the time you will spend amplifying a fragment of 9kb. If you do want to amplify it yourself, I would chop it in smaller portions and clone these together using restriction enzymes... but that's just my opinion.
-dpo-
dpo on Oct 29 2009, 04:51 PM said:
If it's a cDNA from a common species (human, mouse), I would recommend looking for a full length cDNA clone in MGC, Origene, ... or any other company. I think the cost will outweigh the time you will spend amplifying a fragment of 9kb. If you do want to amplify it yourself, I would chop it in smaller portions and clone these together using restriction enzymes... but that's just my opinion.
yeah, cloning in small portions is definitely a better option. in our lab, quite a few genes that we work on, are this big. we hv observed that amplifying it in one go , leads to accumulation of many mutations.....polymerases dont read well for such a long stretch, although there r some new companies which claim that theirs can (forgot the name
)....s> what i wud do, is to amplify and clone in pieces of about 3 kb. all the best!
-DRN-
DRN on Oct 29 2009, 06:12 PM said:
dpo on Oct 29 2009, 04:51 PM said:
If it's a cDNA from a common species (human, mouse), I would recommend looking for a full length cDNA clone in MGC, Origene, ... or any other company. I think the cost will outweigh the time you will spend amplifying a fragment of 9kb. If you do want to amplify it yourself, I would chop it in smaller portions and clone these together using restriction enzymes... but that's just my opinion.
yeah, cloning in small portions is definitely a better option. in our lab, quite a few genes that we work on, are this big. we hv observed that amplifying it in one go , leads to accumulation of many mutations.....polymerases dont read well for such a long stretch, although there r some new companies which claim that theirs can (forgot the name
)....s> what i wud do, is to amplify and clone in pieces of about 3 kb. all the best!
AGREED!!!
-Pradeep Iyer-
Pradeep Iyer on Oct 29 2009, 06:18 PM said:
DRN on Oct 29 2009, 06:12 PM said:
dpo on Oct 29 2009, 04:51 PM said:
If it's a cDNA from a common species (human, mouse), I would recommend looking for a full length cDNA clone in MGC, Origene, ... or any other company. I think the cost will outweigh the time you will spend amplifying a fragment of 9kb. If you do want to amplify it yourself, I would chop it in smaller portions and clone these together using restriction enzymes... but that's just my opinion.
yeah, cloning in small portions is definitely a better option. in our lab, quite a few genes that we work on, are this big. we hv observed that amplifying it in one go , leads to accumulation of many mutations.....polymerases dont read well for such a long stretch, although there r some new companies which claim that theirs can (forgot the name
)....s> what i wud do, is to amplify and clone in pieces of about 3 kb. all the best!
AGREED!!!
I am sorry i should have told that it is of bacterial origin so using cDNA to decrease the effective size of gene wont work. further as Mr pradeep said increasing the conc of polymerase wil help I cud not understand.
If processivity of the polymerase can be affected by the conc of enzyme ? plz let me know .. 
-sud373-
DRN on Oct 29 2009, 06:12 PM said:
dpo on Oct 29 2009, 04:51 PM said:
If it's a cDNA from a common species (human, mouse), I would recommend looking for a full length cDNA clone in MGC, Origene, ... or any other company. I think the cost will outweigh the time you will spend amplifying a fragment of 9kb. If you do want to amplify it yourself, I would chop it in smaller portions and clone these together using restriction enzymes... but that's just my opinion.
yeah, cloning in small portions is definitely a better option. in our lab, quite a few genes that we work on, are this big. we hv observed that amplifying it in one go , leads to accumulation of many mutations.....polymerases dont read well for such a long stretch, although there r some new companies which claim that theirs can (forgot the name
)....s> what i wud do, is to amplify and clone in pieces of about 3 kb. all the best!
Do u mean by using overlapping primers??
-sud373-
sud373 on Oct 30 2009, 12:05 PM said:
Pradeep Iyer on Oct 29 2009, 06:18 PM said:
DRN on Oct 29 2009, 06:12 PM said:
dpo on Oct 29 2009, 04:51 PM said:
If it's a cDNA from a common species (human, mouse), I would recommend looking for a full length cDNA clone in MGC, Origene, ... or any other company. I think the cost will outweigh the time you will spend amplifying a fragment of 9kb. If you do want to amplify it yourself, I would chop it in smaller portions and clone these together using restriction enzymes... but that's just my opinion.
yeah, cloning in small portions is definitely a better option. in our lab, quite a few genes that we work on, are this big. we hv observed that amplifying it in one go , leads to accumulation of many mutations.....polymerases dont read well for such a long stretch, although there r some new companies which claim that theirs can (forgot the name
)....s> what i wud do, is to amplify and clone in pieces of about 3 kb. all the best!
AGREED!!!
I am sorry i should have told that it is of bacterial origin so using cDNA to decrease the effective size of gene wont work. further as Mr pradeep said increasing the conc of polymerase wil help I cud not understand.
If processivity of the polymerase can be affected by the conc of enzyme ? plz let me know .. hey no.......increasing the conc. of polymerase is not gonna help!!! going above the optimal level doesn't increase the efficiency of an enzyme.
-DRN-
DRN on Oct 30 2009, 01:55 PM said:
hey no.......increasing the conc. of polymerase is not gonna help!!! going above the optimal level doesn't increase the efficiency of an enzyme.
Ya i am aware of that.. what i meant was if it is not optimum cause then it will get exhausted!!! may be i was not clear
and i still stick to the dNTP's in excess
-Pradeep Iyer-