11 replies to this topic

[sud373]

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??

[Prep!]

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??

Well by increasing the extension time!!!
and add may be more polemerase than usual so that it does not get exhausted in between!!!

[Prep!]

not to forget dNTPs too in excess!!!

[dpo]

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.

[Vini]

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!  

[Prep!]

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!!!

[sud373]

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??

[Vini]

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.

[Prep!]

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

[Vini]

Pradeep Iyer, on Oct 30 2009, 03:14 PM, said:

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

yeah, u might stick to ur stance on dNTPs in excess, but polymerase might not stick to its template.....point of disagreement, eh????   ......lack of clarity this time also, i guess!  

[phage434]

I have had very good luck with the Roche Expand long range PCR system, and would certainly try it long before doing the work of splicing together many fragments from multiple PCR reactions.  PCRs up to 20 Kb are routine.