DNA ladder is at wrong MW's

[breathoffire]

Okay, I ran a 1% TBE gel and nothing is smeared or odd looking ....but my ladder is very off from the image that is on the companies website. Also the website said that the most intense band is a 500bp and you can clearly see that the lower intensity bands are more intensy.

[Sandra_T]

Hi Breathoffire. The modern molecular markers are not robust, especially if you are staining in-gel and then running with the stain in the gel and deviate at all from the manufacturers instructions. If you want to quantify at all, then I personally would recommend back to basics and use Lamba DNA cut with HindIII, so that you know that you are not looking at some companies artifact! I wasted a lot of time with some of these commercial markers... To get reliable reproducible staining to quantify, best to run the gel without stain and then shake the gel gently with TBE and stain after running the gel. To ensure that the gel is good quality, make sure you boil the agarose a good couple of times and pour reasonably hot (meaning so you can touch the glass with your hand but only for a few seconds!)

[Bonnie]

Can you post a link to the company's picture of what the ladder is supposed to look like? That does seem pretty weird.

[breathoffire]

Originally Posted by Bonnie

Can you post a link to the company's picture of what the ladder is supposed to look like? That does seem pretty weird.

Here's the company's website.
htt p://ww w.neb.com/ nebecomm/ products/prod uctN0467.asp

[breathoffire]

Originally Posted by Sandra_T

Hi Breathoffire. The modern molecular markers are not robust, especially if you are staining in-gel and then running with the stain in the gel and deviate at all from the manufacturers instructions. If you want to quantify at all, then I personally would recommend back to basics and use Lamba DNA cut with HindIII, so that you know that you are not looking at some companies artifact! I wasted a lot of time with some of these commercial markers... To get reliable reproducible staining to quantify, best to run the gel without stain and then shake the gel gently with TBE and stain after running the gel. To ensure that the gel is good quality, make sure you boil the agarose a good couple of times and pour reasonably hot (meaning so you can touch the glass with your hand but only for a few seconds!)

This was really helpful. I just started at a new lab where they stain in-gel. Previously, I had only done out of gel staining so this really stumped me, because this is something I have never seen before.
Also, I didn't know that you could use lambda cut with HindI III. so thanks I learned something new.