11 replies to this topic

[xyz]

Hi,
I am doing the cloning experiment. SO i have amplified my product thru PCR and after that visualized it thru 1% agarose gel. Now i want to run on gel ( with large combo size ) so that I can purify it. I want to know the volume of PCr product and DNA ladder required to load in a gel

[bob1]

volume depends on the concentration of your products.  The higher concentration, the less you need to add.  Also, the bigger your well in width, the more you you need to add.  For a PCR product that you are trying to clean up, I would load as much as you can get.

[jiajia1987]

For purification of PCR products via gel extraction, I always load everything.

[aztecan princess]

It hard to say, it depend or how much amplification you have. If you have a very good amplification, you can run 5-10 ul of your PCR reaction, better if you load everything. Use (again depend of the ladder you are using) for example 5 ul of 0.1 ug/ul ladder.
And very important it depend the kit you are using to purify your DNA, take account of the specification in the kit. For example, for QIAaquik  kit: use up to 400 mg of agarose fragment, if you use more, you’ll recover very few DNA. Load up to 10 ug DNA/column, if you have more DNA than that, use two columns.
In few words, READ THE KIT'S MANUAL BEFORE DO IT.

[xyz]

Hi,
Thanks for information, but I am naive in molecular world, so want to know that how would I estimate the concentration of my PCR product(DNA)

aztecan princess, on Jun 2 2009, 07:41 AM, said:

It hard to say, it depend or how much amplification you have. If you have a very good amplification, you can run 5-10 ul of your PCR reaction, better if you load everything. Use (again depend of the ladder you are using) for example 5 ul of 0.1 ug/ul ladder.
And very important it depend the kit you are using to purify your DNA, take account of the specification in the kit. For example, for QIAaquik  kit: use up to 400 mg of agarose fragment, if you use more, you’ll recover very few DNA. Load up to 10 ug DNA/column, if you have more DNA than that, use two columns.
In few words, READ THE KIT'S MANUAL BEFORE DO IT.

[xyz]

Hi,
Thanks
Means for a bigger well 30-35[ul] is sufficient? and how would i know the concentration of my product.[/u]

bob1, on Jun 1 2009, 05:26 PM, said:

volume depends on the concentration of your products.  The higher concentration, the less you need to add.  Also, the bigger your well in width, the more you you need to add.  For a PCR product that you are trying to clean up, I would load as much as you can get.

[microgirl]

I generally just go ahead and run 10 ul per well (5 if I'm really trying not to use up my pcr product) for a 12-well comb in a mini gel. The promega ladder that we use calls for 5 ul. I add about 3 ul of loading dye. Just go ahead and try it - you can always make more pcr product if you need to.

xyz, on Jun 2 2009, 09:21 AM, said:

Hi,
Thanks
Means for a bigger well 30-35[ul] is sufficient? and how would i know the concentration of my product.[/u]

bob1, on Jun 1 2009, 05:26 PM, said:

volume depends on the concentration of your products.  The higher concentration, the less you need to add.  Also, the bigger your well in width, the more you you need to add.  For a PCR product that you are trying to clean up, I would load as much as you can get.

[dgclone]

In short compare to a standard in your ladder.

Your ladder will have a couple of bands that will probably be brighter in intensity than the others. These are often bands of known concentration and can be used to estimate the concentration of your band. Ie if your band is equally as bright as your ladder band then you have the same concentration. Most often you just have to guess and remember to include both your dilution factor in your calculation as well as adjust to your final PCR volume

xyz, on Jun 2 2009, 09:19 AM, said:

Hi,
Thanks for information, but I am naive in molecular world, so want to know that how would I estimate the concentration of my PCR product(DNA)

aztecan princess, on Jun 2 2009, 07:41 AM, said:

It hard to say, it depend or how much amplification you have. If you have a very good amplification, you can run 5-10 ul of your PCR reaction, better if you load everything. Use (again depend of the ladder you are using) for example 5 ul of 0.1 ug/ul ladder.
And very important it depend the kit you are using to purify your DNA, take account of the specification in the kit. For example, for QIAaquik  kit: use up to 400 mg of agarose fragment, if you use more, you’ll recover very few DNA. Load up to 10 ug DNA/column, if you have more DNA than that, use two columns.
In few words, READ THE KIT'S MANUAL BEFORE DO IT.

[jiajia1987]

xyz, on Jun 3 2009, 12:21 AM, said:

Hi,
Thanks
Means for a bigger well 30-35[ul] is sufficient? and how would i know the concentration of my product.[/u]

bob1, on Jun 1 2009, 05:26 PM, said:

volume depends on the concentration of your products.  The higher concentration, the less you need to add.  Also, the bigger your well in width, the more you you need to add.  For a PCR product that you are trying to clean up, I would load as much as you can get.

if you want to know the concentration of your PCR products, you can always make use of a nanodrop, or a spectrophotometer.

[xyz]

Thanks for information

microgirl, on Jun 2 2009, 11:35 AM, said:

I generally just go ahead and run 10 ul per well (5 if I'm really trying not to use up my pcr product) for a 12-well comb in a mini gel. The promega ladder that we use calls for 5 ul. I add about 3 ul of loading dye. Just go ahead and try it - you can always make more pcr product if you need to.

xyz, on Jun 2 2009, 09:21 AM, said:

Hi,
Thanks
Means for a bigger well 30-35[ul] is sufficient? and how would i know the concentration of my product.[/u]

bob1, on Jun 1 2009, 05:26 PM, said:

volume depends on the concentration of your products.  The higher concentration, the less you need to add.  Also, the bigger your well in width, the more you you need to add.  For a PCR product that you are trying to clean up, I would load as much as you can get.

[bob1]

jiajia1987, on Jun 3 2009, 01:47 AM, said:

if you want to know the concentration of your PCR products, you can always make use of a nanodrop, or a spectrophotometer.

Only if you have cleaned them up, free dNTPs interfere with this reading.

[jiajia1987]

bob1, on Jun 4 2009, 08:06 AM, said:

jiajia1987, on Jun 3 2009, 01:47 AM, said:

if you want to know the concentration of your PCR products, you can always make use of a nanodrop, or a spectrophotometer.

Only if you have cleaned them up, free dNTPs interfere with this reading.

yep. either by DNA cleanup or gel extraction.