IGEM:Harvard/2006/DNA nanostructures/Notebook/2006-8-23: Difference between revisions
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== PEG fractionation (from [[IGEM:Harvard/2006/DNA_nanostructures/Notebook/2006-8-20|8-20]]) == | == PEG fractionation (from [[IGEM:Harvard/2006/DNA_nanostructures/Notebook/2006-8-20|8-20]]) == | ||
Also including 8% and 10% PEG fractionation for c6.0.A, c6.0.B, and c6.0.C | Also including 8% and 10% PEG fractionation for c6.0.A, c6.0.B, and c6.0.C | ||
==Streptavidin Depletion Assay== | |||
===Plans=== | |||
*Possible Issues and Solutions | |||
**Too much DNA; even if a reasonable amount (say 70% of the biotinylated sites) are binding strep, there wouldn't be a noticeable difference between the bead-treated boxes and non-bead-treated boxes. | |||
***Lower amount of DNA used to half a reaction. | |||
****But the bands aren't that bright as it is, despite the iris being completely open, manual exposure, and use of 5uL of EtBr - possibly due to DNA leeching out during the long (>2hour) electrophoresis? Is that likely at all? But at the same time, my gels have been coming out fainter and fainter as I run longer each time, and yesterday it wasn't even left in buffer for any amount of time at all. | |||
*****Run at higher voltage (100V), shorter time (1.5hr) - the difficulty in differentiating the folded from the scaffold might be due recently to the switchover to 0.5xTBE. Take a risk with the higher voltage - check on the gel box every 20 minutes to make sure no boiling is occuring, and scrape off the electrode wire each time. | |||
**Too little free streptavidin | |||
***Overload with free streptavidin - add from the original stock tube, which is likely safer (in more accurate-for-streptavidin-efficacy) buffer than the dilutions that have been made. This should push the equilibrium towards more complete binding of sites. | |||
**Biotinylated stuff not binding. | |||
***Check by running a control of biotinylated oligos (untreated, beaded, free streped, free then beaded) - there are large amounts of the original tube oligos, so run 40pmol of biotinylated sites (10uM biotinylated sites in the pre-working stock = 10pmoles/uL), or 5uL (to account for inefficient binding and pipetting error) in one lane and 40uL in another, just to make sure that the binding actually works. | |||
****If oligos don't show up in the untreated lane, try SYBR gold staining - ssDNA will show up better at low concentrations that way. | |||
**Suspicion that the magnetic beads may work better because there's no worry of accidentally loading part of the pellet and, thus, bead-bound material. | |||
***Use magnetic beads, concentrated with the MagnaRack initially, and run high concentrations (ie. low total volumes). | |||
===Folded:=== | |||
*4 rxns each of: | |||
**c5.0.A lidless | |||
**c5.0.Eb | |||
**(4 rxns of c5.0.Fb remain from Katie's foldings on Monday) | |||
*NB: Noticed that there were errors in the pre-working stock table. See [[Table_of_c5.0_Working_Stocks|c5.0 Table of Working Stocks]]. | |||
**Had to mix: | |||
***c5.0.Eb | |||
***c5.0.Fb |
Revision as of 18:19, 23 August 2006
Verify folding of design 5 and 6
Ran gel to verify folding from earlier this week. Also comparing new scaffold with old scaffold.
Lane | Contents |
1 | ladder |
2 | p7308 old |
3 | p7308 new |
4 | c5.0.A (old scaffold) |
5 | c5.0.A (new scaffold) |
6 | c5.0.C |
7 | c5.0.D |
8 | c6.0.A |
9 | c6.0.B |
10 | c6.0.C |
PEG fractionation (from 8-20)
Also including 8% and 10% PEG fractionation for c6.0.A, c6.0.B, and c6.0.C
Streptavidin Depletion Assay
Plans
- Possible Issues and Solutions
- Too much DNA; even if a reasonable amount (say 70% of the biotinylated sites) are binding strep, there wouldn't be a noticeable difference between the bead-treated boxes and non-bead-treated boxes.
- Lower amount of DNA used to half a reaction.
- But the bands aren't that bright as it is, despite the iris being completely open, manual exposure, and use of 5uL of EtBr - possibly due to DNA leeching out during the long (>2hour) electrophoresis? Is that likely at all? But at the same time, my gels have been coming out fainter and fainter as I run longer each time, and yesterday it wasn't even left in buffer for any amount of time at all.
- Run at higher voltage (100V), shorter time (1.5hr) - the difficulty in differentiating the folded from the scaffold might be due recently to the switchover to 0.5xTBE. Take a risk with the higher voltage - check on the gel box every 20 minutes to make sure no boiling is occuring, and scrape off the electrode wire each time.
- But the bands aren't that bright as it is, despite the iris being completely open, manual exposure, and use of 5uL of EtBr - possibly due to DNA leeching out during the long (>2hour) electrophoresis? Is that likely at all? But at the same time, my gels have been coming out fainter and fainter as I run longer each time, and yesterday it wasn't even left in buffer for any amount of time at all.
- Lower amount of DNA used to half a reaction.
- Too little free streptavidin
- Overload with free streptavidin - add from the original stock tube, which is likely safer (in more accurate-for-streptavidin-efficacy) buffer than the dilutions that have been made. This should push the equilibrium towards more complete binding of sites.
- Biotinylated stuff not binding.
- Check by running a control of biotinylated oligos (untreated, beaded, free streped, free then beaded) - there are large amounts of the original tube oligos, so run 40pmol of biotinylated sites (10uM biotinylated sites in the pre-working stock = 10pmoles/uL), or 5uL (to account for inefficient binding and pipetting error) in one lane and 40uL in another, just to make sure that the binding actually works.
- If oligos don't show up in the untreated lane, try SYBR gold staining - ssDNA will show up better at low concentrations that way.
- Check by running a control of biotinylated oligos (untreated, beaded, free streped, free then beaded) - there are large amounts of the original tube oligos, so run 40pmol of biotinylated sites (10uM biotinylated sites in the pre-working stock = 10pmoles/uL), or 5uL (to account for inefficient binding and pipetting error) in one lane and 40uL in another, just to make sure that the binding actually works.
- Suspicion that the magnetic beads may work better because there's no worry of accidentally loading part of the pellet and, thus, bead-bound material.
- Use magnetic beads, concentrated with the MagnaRack initially, and run high concentrations (ie. low total volumes).
- Too much DNA; even if a reasonable amount (say 70% of the biotinylated sites) are binding strep, there wouldn't be a noticeable difference between the bead-treated boxes and non-bead-treated boxes.
Folded:
- 4 rxns each of:
- c5.0.A lidless
- c5.0.Eb
- (4 rxns of c5.0.Fb remain from Katie's foldings on Monday)
- NB: Noticed that there were errors in the pre-working stock table. See c5.0 Table of Working Stocks.
- Had to mix:
- c5.0.Eb
- c5.0.Fb
- Had to mix: