I need to run gels that are of not the "most" importance. So I do not want to waste alot of money on step ladder. How do I dilute DNA step ladder? Is there a general procedure, for I have tried googling for one, but I get varying answers, such as a 1:4 dilution, and so on.

This is the information on the Step ladder, by Promega, posted as an image, since copy and pasting would mess up readability.

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Even though I awarded the bounty because it gave me the same concept; what I had actually did was to assume I would use $60ul$ of ladder combined with $1$x dye.

Therefore since I started with $1ml$ of $6$x loading dye, $100ul$ of DNA step ladder, and available nuclease free $\ce{H2O}$, and I desired a 5:1 ratio of solution(dye and $\ce{H2O}$) to ladder.

I would then use $12ul$ of stock to the $60ul$ total.

To make the dye $1$x, I would add $10ul$ of the $6$x dye to make the final concenetration of dye $1$x.

Then I would add up to $38ul$ with nuclease free $\ce{H2O}$.

So, sorry if this was worded redundantly, but It helps me to read, therefore in summary:

$$ +10ul \text{ of 6x dye}$$ $$ +12ul \text{ of stock 100bp DNA step Ladder}$$ $$+ 38ul \text{ Nuclease Free } \ce{H2O} $$ $$ \text{__________________________________}$$

$$\sum{60ul} \text{ of ladder & dye} (1 \text{x}) $$

of which total I can aliquot out to my hearts desire.

EDIT2: The ladder turned out successful.


You'll probably have to titrate it down yourself. You might be able to estimate: The detection limit for ethidium bromide staining is about $1 ng$ per band. The insert says it's at "1 $\mu g/ \mu l$", but that's for all 40 bands and they're clearly not all at the same concentration. Depending on which bands you care about (1600 and below are dim), you might be able to get away with $(\frac{1}{25})^{th}$ their recommended concentration (1 $\mu g / 40$ bands = 25 $ng$) -- but I would titrate a small amount and see what works.

  • $\begingroup$ I am trying to visualize the mitochondrial cytochrome b gene of D. melanogaster if that is of any help in this endeavor. I dont think its a larger region according to Flybase. flybase.org/reports/FBgn0013678.html But to be honest I am a novice. $\endgroup$
    – Ro Siv
    Nov 15 '15 at 3:03
  • $\begingroup$ That gene is ~1 kb, and the ladder is maybe 1/4 as bright in that range compared to the HMW bands. Try diluting around 1:5 (add 4 ul of TE buffer or water per 1 ul of ladder). $\endgroup$
    – ZachB
    Nov 16 '15 at 5:51

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