7 replies to this topic

[bigvels]

I need to clone one gene from Gram-positive bacteria into E. coli using pET-15b vector. Now, I have to digest them with Fast digest ® BamHI and XhoI enzymes (Fermentas). In Fermentas manual, it was mentioned that these Fast digest enzymes will cut the DNA in 15 min. Once, I subjected digestion for 4 hours at 37⁰C. For, pET 15b, I observed several bands. Can anyone help me, how much exact time I need to digest using Fast digest (Fermentas) BamHI and XhoI?

[swanny]

bigvels, on Jan 18 2010, 02:44 AM, said:

I need to clone one gene from Gram-positive bacteria into E. coli using pET-15b vector. Now, I have to digest them with Fast digest ® BamHI and XhoI enzymes (Fermentas). In Fermentas manual, it was mentioned that these Fast digest enzymes will cut the DNA in 15 min. Once, I subjected digestion for 4 hours at 37⁰C. For, pET 15b, I observed several bands. Can anyone help me, how much exact time I need to digest using Fast digest (Fermentas) BamHI and XhoI?

I've always had success using the FastDigest enzymes, and typically do a 1hr digest.

Do you have an image of the gel for us? Or else, what are the band sizes you come up with? Are any of them the expected size?

[bigvels]

swanny, on Jan 17 2010, 09:02 PM, said:

bigvels, on Jan 18 2010, 02:44 AM, said:

I need to clone one gene from Gram-positive bacteria into E. coli using pET-15b vector. Now, I have to digest them with Fast digest ® BamHI and XhoI enzymes (Fermentas). In Fermentas manual, it was mentioned that these Fast digest enzymes will cut the DNA in 15 min. Once, I subjected digestion for 4 hours at 37⁰C. For, pET 15b, I observed several bands. Can anyone help me, how much exact time I need to digest using Fast digest (Fermentas) BamHI and XhoI?

I've always had success using the FastDigest enzymes, and typically do a 1hr digest.

Do you have an image of the gel for us? Or else, what are the band sizes you come up with? Are any of them the expected size?

Thanks Swanny for your reply.
I don't have any picture. I have not saved it. I have bands in the region between 3 kb to 5.5 kb. One was at expected size of 5.5 kb region. Can I do double digestion (Addition of both BamHI and XhoI in the same reaction mix using universal Fast digestion buffer) using both Fast digest enzymes (Fermentas) for my 1.6 kb insert as well as pET 15b for a period of 1hr? Will it work for my case? Hope it will work for me. How is your experience in cloning using Fast Digest (Fermentas) enzymes? Do you have any failure?

[swanny]

bigvels, on Jan 19 2010, 11:51 AM, said:

swanny, on Jan 17 2010, 09:02 PM, said:

bigvels, on Jan 18 2010, 02:44 AM, said:

I need to clone one gene from Gram-positive bacteria into E. coli using pET-15b vector. Now, I have to digest them with Fast digest ® BamHI and XhoI enzymes (Fermentas). In Fermentas manual, it was mentioned that these Fast digest enzymes will cut the DNA in 15 min. Once, I subjected digestion for 4 hours at 37⁰C. For, pET 15b, I observed several bands. Can anyone help me, how much exact time I need to digest using Fast digest (Fermentas) BamHI and XhoI?

I've always had success using the FastDigest enzymes, and typically do a 1hr digest.

Do you have an image of the gel for us? Or else, what are the band sizes you come up with? Are any of them the expected size?

Thanks Swanny for your reply.
I don't have any picture. I have not saved it. I have bands in the region between 3 kb to 5.5 kb. One was at expected size of 5.5 kb region. Can I do double digestion (Addition of both BamHI and XhoI in the same reaction mix using universal Fast digestion buffer) using both Fast digest enzymes (Fermentas) for my 1.6 kb insert as well as pET 15b for a period of 1hr? Will it work for my case? Hope it will work for me. How is your experience in cloning using Fast Digest (Fermentas) enzymes? Do you have any failure?

Double digestion should work perfectly, because that is one of the aims of the FastDigest system. Follow the protocols exactly with both single digests as well as your double digest. I've not had any issues using FastDigest enzymes.

[bigvels]

swanny, on Jan 18 2010, 07:25 PM, said:

bigvels, on Jan 19 2010, 11:51 AM, said:

swanny, on Jan 17 2010, 09:02 PM, said:

bigvels, on Jan 18 2010, 02:44 AM, said:

I need to clone one gene from Gram-positive bacteria into E. coli using pET-15b vector. Now, I have to digest them with Fast digest ® BamHI and XhoI enzymes (Fermentas). In Fermentas manual, it was mentioned that these Fast digest enzymes will cut the DNA in 15 min. Once, I subjected digestion for 4 hours at 37⁰C. For, pET 15b, I observed several bands. Can anyone help me, how much exact time I need to digest using Fast digest (Fermentas) BamHI and XhoI?

I've always had success using the FastDigest enzymes, and typically do a 1hr digest.

Do you have an image of the gel for us? Or else, what are the band sizes you come up with? Are any of them the expected size?

Thanks Swanny for your reply.
I don't have any picture. I have not saved it. I have bands in the region between 3 kb to 5.5 kb. One was at expected size of 5.5 kb region. Can I do double digestion (Addition of both BamHI and XhoI in the same reaction mix using universal Fast digestion buffer) using both Fast digest enzymes (Fermentas) for my 1.6 kb insert as well as pET 15b for a period of 1hr? Will it work for my case? Hope it will work for me. How is your experience in cloning using Fast Digest (Fermentas) enzymes? Do you have any failure?

Double digestion should work perfectly, because that is one of the aims of the FastDigest system. Follow the protocols exactly with both single digests as well as your double digest. I've not had any issues using FastDigest enzymes.

Hi Swanny
Which one you prefer sequencial single digestion or double digestion for 1 hr. Can you give your protocol of restriction digestion to me? I will follow the same as you standardised it. Give the protocol as well as ur suggestion.

[swanny]

OK, this is straight from the Fast Digest data sheets.


                        Plasmid DNA     PCR             Genomic DNA
Water                    15                17                   30
10x FD buffer           2                  3                    5
DNA                        2 (to 1ug)    10 (~.2ug)       10 (~5 ug)
FD enzyme              1                  1                     5

for double digests, scale up reaction conditions or plasmid DNA especially to 30ul. Add 1ul each enzyme. Make sure volume of all enzymes is less than 1/10 total volume.

Incubate 5-15 minutes. I have digested 1 ug to up to 5ug in a reaction, simply by adding more enzyme.

Edited by swanny, 18 January 2010 - 08:54 PM.

[bigvels]

swanny, on Jan 18 2010, 08:53 PM, said:

OK, this is straight from the Fast Digest data sheets.


                        Plasmid DNA     PCR             Genomic DNA
Water                    15                17                   30
10x FD buffer           2                  3                    5
DNA                        2 (to 1ug)    10 (~.2ug)       10 (~5 ug)
FD enzyme              1                  1                     5

for double digests, scale up reaction conditions or plasmid DNA especially to 30ul. Add 1ul each enzyme. Make sure volume of all enzymes is less than 1/10 total volume.

Incubate 5-15 minutes. I have digested 1 ug to up to 5ug in a reaction, simply by adding more enzyme.

Thanks Swanny for ur reply.
I have one small doubt. You told me that you got the success at incubation for 1 hr. Now in protocol, you wrote to me that the incubation time will be 5 - 15 min. Please confirm it and tell me sothat quickly I will give a try. I have to get single band of both Plasmid vector as well as PCR. PLease also write to me about other troubleshoots also.
Thanks.

Edited by bigvels, 19 January 2010 - 06:24 AM.

[swanny]

I have never done a complete time-trial of digestion. The protocol says 5-15 min; give it a go and see what you get, as well as doing the hour digest.

It's a bit hard to give a meaningful troubleshooting list. try the digesions we've talked about, get an image to upload and we can discuss further if there are issues. Sometimes the only thing to do is try the experiment, as it's the best way to learn.