5 replies to this topic

[xyz]

Hi all,
I have purified my PCR product by QiaexII kit and by eye visualization I guees that the  concentration of product is around 4[ug] in 40[ul] in total volume. Now I want to digest this PCR product and vector that already have any other gene in it and I want to replace it with my PCR product. I had BAMH1 restriction sites in PCR prodct and vector. So what to do for digestion? What concentration of enzyme,PCR product ,BSA should be used both for PCR product and vector. And how much final volume should I use?
Its really giving me a lot of headache to decide everything. Need help

[hanming86]

xyz, on Jun 3 2009, 07:52 AM, said:

Hi all,
I have purified my PCR product by QiaexII kit and by eye visualization I guees that the  concentration of product is around 4[ug] in 40[ul] in total volume. Now I want to digest this PCR product and vector that already have any other gene in it and I want to replace it with my PCR product. I had BAMH1 restriction sites in PCR prodct and vector. So what to do for digestion? What concentration of enzyme,PCR product ,BSA should be used both for PCR product and vector. And how much final volume should I use?
Its really giving me a lot of headache to decide everything. Need help

It shoudln't be giving u headache.. seriously

just read the manual. follow the instruction . u be fine.
BamHi can't be heat inactivated efficiently.

I really don't know why this can give u headache.. . . .

[xyz]

Because I am not able to decide the concentrations at all, so-------------. I read the manual but still I have some confusions and I am not sure about vector digestion?We use the biolab product for digestion. do u have experience for that?

hanming86, on Jun 3 2009, 09:44 AM, said:

xyz, on Jun 3 2009, 07:52 AM, said:

Hi all,
I have purified my PCR product by QiaexII kit and by eye visualization I guees that the  concentration of product is around 4[ug] in 40[ul] in total volume. Now I want to digest this PCR product and vector that already have any other gene in it and I want to replace it with my PCR product. I had BAMH1 restriction sites in PCR prodct and vector. So what to do for digestion? What concentration of enzyme,PCR product ,BSA should be used both for PCR product and vector. And how much final volume should I use?
Its really giving me a lot of headache to decide everything. Need help

It shoudln't be giving u headache.. seriously

just read the manual. follow the instruction . u be fine.
BamHi can't be heat inactivated efficiently.

I really don't know why this can give u headache.. . . .

[hanming86]

What's your confusion and what's ur approach?

Tell us ur reaction . I want you to come up with something. then we will look at the problem.

Vector digestion and PCR product digestion share the same reaction. nothing special. just use the standard reaction condition..

There's really no FIXED thing around here. Some ppl like to use more some swear to using less of the same compound.

BSA conc. is normally written on the manual so i don't know why u ask this.
BamHI just use 1ul for whatever .

You can't determine the  concentration at all so what? just run the digestion as usual. 1U = 1ug. but 10 fold of that is still fine

Seriously just run the reaction . u will feel better.

regards,

Ming

[xyz]

Thanks
I had run the digestion and made the total voume 30[ul] , so i can directly run it on gel. I have one more question that how much amount of DNA is required for digestion? is it 1 microgram?
Thanks again

hanming86, on Jun 3 2009, 08:45 PM, said:

What's your confusion and what's ur approach?

Tell us ur reaction . I want you to come up with something. then we will look at the problem.

Vector digestion and PCR product digestion share the same reaction. nothing special. just use the standard reaction condition..

There's really no FIXED thing around here. Some ppl like to use more some swear to using less of the same compound.

BSA conc. is normally written on the manual so i don't know why u ask this.
BamHI just use 1ul for whatever .

You can't determine the  concentration at all so what? just run the digestion as usual. 1U = 1ug. but 10 fold of that is still fine

Seriously just run the reaction . u will feel better.

regards,

Ming

[jiajia1987]

Usually, I will digest 5micrograms of my vector and purify it after that. As for PCR products, I will digest perhaps 5 microliters depending on the concentration of PCR products I wish to digest. Like what haming89 said, it all depends on what you want. DIfferent people have different ways of doing it.