Daniel:Notebook/HiResChrPaint/2013-7-12: Difference between revisions
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Summary: '''A''': Optimal labeling ratio for ULS dyes is around 2%, or 1 in 50 bp. This experiment was done on Alexa 488 so it should be directly applicable to my research. '''B''': Increasing reaction time up to 1 hour should only improve labeling. Optimal time is suggested around 15 minutes but I think 20-30 wouldn't be too bad either. '''C''': | Summary: '''A''': Optimal labeling ratio for ULS dyes is around 2%, or 1 in 50 bp. This experiment was done on Alexa 488 so it should be directly applicable to my research. '''B''': Increasing reaction time up to 1 hour should only improve labeling. Optimal time is suggested around 15 minutes but I think 20-30 wouldn't be too bad either. '''C''': Using more dye improves labeling efficiency, rounding off at around 1.4x dye:DNA. This is expected, but might have been an issue if the dye reacted with itself. '''D''': Optimal temperature for the reaction is between 70 and 80 C. Also expected, and not an issue in my protocols. | ||
Basically this tells me that as I expected, adding more dye and incubating longer should get me better yields. Also of note, reaction temperature is fine and I'm shooting for around 50 bp per dye molecule. The [[File:ULYSIS_Manual.pdf|ULYSIS Manual]] suggests a range of 43 to 67 bases per dye molecule, so this is about right. |
Revision as of 18:12, 12 July 2013
S2 Probe Prep (Started 5/29/2013)
I've been performing some research on the ULS labeling system to see if I can get a good grasp on what's going on before I try labeling again. The most telling, I think, is some figures from a poster I got on Invitrogen's webstite.
Research Notes: ULS
A | B |
File:ULS-LabelingRatio.png | File:ULS-ReactionDuration.png |
C | D |
File:ULS-ReactionRatio.png | File:ULS-ReactionTemp.png |
Summary: A: Optimal labeling ratio for ULS dyes is around 2%, or 1 in 50 bp. This experiment was done on Alexa 488 so it should be directly applicable to my research. B: Increasing reaction time up to 1 hour should only improve labeling. Optimal time is suggested around 15 minutes but I think 20-30 wouldn't be too bad either. C: Using more dye improves labeling efficiency, rounding off at around 1.4x dye:DNA. This is expected, but might have been an issue if the dye reacted with itself. D: Optimal temperature for the reaction is between 70 and 80 C. Also expected, and not an issue in my protocols.
Basically this tells me that as I expected, adding more dye and incubating longer should get me better yields. Also of note, reaction temperature is fine and I'm shooting for around 50 bp per dye molecule. The ULYSIS Manual suggests a range of 43 to 67 bases per dye molecule, so this is about right.