DNA Maxi Preparation - Viscous DNA? (Aug/26/2008 )

hallo,

When i dissolve my DNA pellet at the end of the preparation, it results in a very viscous sample.
what could be the reason for this?
I want to inject my DNA into mice, therefore i want to get sure that DNA is clean.

---

What volume are you resuspending in, you may just have loads of material. How are you doing the prep ? What does your sample look like on a gel ?

---

thanks for reply.

i resuspended in 500µl H2O and this results in 2,3µg/µl DNA.
i don´t have run a gel until now.
i usually use kit from macherey nagel.

could this be protein? but normally you should get rid of protein with this kit?

---

You really should run it on a gel not only to see that it is clean, free of RNA and that all three conformations are present but also to confirm the concentration you measured before you do anything with it. I would do some dilutions and run it alongside a good marker of known concentration.

---

2,3 µg/µl should not be so viscous, I use 1 µg/µl and its like water, I also use the MN kits for maxi prep. Jenny is right, run a gel and take a look at the DNA to see if its clean and if the concetration is the same as the one your photometer says.

---

i will run a gel.

but could there be also some protein contamination?

A260 0,465
A280 0,309

A260/280 ratio = 1,51

---

For DNA 260/280 ratio should be higher than 1.7 to be sure you don't have protein contamination.

---

I will make a phenol-chloroform-extraction and check DNA again.

...but what could be the reason for protein contamination during?

---

Phenol-chlorophorm extraction needs a very pristine process (you know, taking the supernatant carefully enough to not taking the protein clumps, taking phenol from the bottom phase only, heat treatment, careful while handling the recently centrifugated tubes... this can go forever... so be careful while doing that!

On the other hand, after using maxi prep kits usually you will get a pretty clean sample, but if you have way too much DNA (big pellet, for instance) you might need to use more DDW to dissolve it (and if this affect your DNA concentration you can always do isopropanol precipitation (actually I always do that after DNA extraction, but that depends of how much DNA you need).

Anyway, I am new in this so probably some experienced people can help you here with this.

I am not familiar with the kit you are using (I use GerardBiotech Maxi prep kit) but at the end of the protocol booklet usually there are some troubleshooting topics, check if there's something regarding protein contamination.

---