Haynes Lab:Notebook/Investigating Photo-Switchable Synthetic Nucleosomes/2012/11/21: Difference between revisions
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==11/21/12== | ==11/21/12== | ||
'''Results from yesterday's ligation''' | |||
* Got 1:1 ratio of ligation: negative control for both | |||
* About 10 colonies total on every plate | |||
* Forgot to take into account X/S vector self-ligation | |||
* Will pick 4 colonies from each (H2B, LOV) to see if anything worked. | |||
* In the mean time, will retry cloning, trouble shoot procedure with help from Dr. Haynes | |||
---- | |||
* Repeat PCR of H2B and LOV inserts | * Repeat PCR of H2B and LOV inserts | ||
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'''Assemblies''' | '''Assemblies''' | ||
# H2B - X/S - 410 bp + pSB1A3 - X/S - 2000 bp | # NEB T4 ligase; H2B - X/S - 410 bp + pSB1A3 - X/S - 2000 bp | ||
# NEB T4 ligase; LOV - X/S - 461 bp + pSB1A3 - X/S - 2000 bp | |||
# NEB T4 ligase; (negtaive control) pSB1A3 - X/S - 2000 bp | |||
# Roche T4 ligase; H2B - X/S - 410 bp + pSB1A3 - X/S - 2000 bp | |||
# Roche T4 ligase; LOV - X/S - 461 bp + pSB1A3 - X/S - 2000 bp | |||
# Roche T4 ligase; (negtaive control) pSB1A3 - X/S - 2000 bp | |||
* Digests: | * Digests: | ||
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| Sample|| 260|| 260/280 || ng/μL | | Sample|| 260|| 260/280 || ng/μL | ||
|- | |- | ||
| H2B - X/S || | | H2B - X/S 1 || 0.016 ||1.807|| 15.961 | ||
|- | |- | ||
| LOV - X/S | | H2B - X/S 2 || 0.009 || 1.976 || 8.619 | ||
|- | |||
| LOV - X/S 1 || 0.006|| 1.694 || 6.455 | |||
|- | |||
| LOV - X/S 2 || 0.025|| 1.883 || 25.363 | |||
|} | |} | ||
[[Image:VN_Gel_11- | [[Image:VN_Gel_11-21-12.png|Results of from the digests are shown.]] | ||
[[Image:KAH_Fermentas_GeneRuler_1kbplus.jpg]] | [[Image:KAH_Fermentas_GeneRuler_1kbplus.jpg]] | ||
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# LOV = 461 | # LOV = 461 | ||
* There were two samples made of each PCR reaction. The two from the H2B are shown on the left, and the two from the LOV are shown on the right. | |||
'''Dephosphorylation''' | '''Dephosphorylation''' (11/23/12) | ||
> Dephosphorylation (Roche) | > Dephosphorylation (Roche) | ||
# pSB1A3 - X/S = | # pSB1A3 - X/S = 2000 bp | ||
{| class="wikitable" border="0" cellspacing="3" <!-- Dephos table --> | {| class="wikitable" border="0" cellspacing="3" <!-- Dephos table --> | ||
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| <u>Reagent</u> || <u>Volume</u> | | <u>Reagent</u> || <u>Volume</u> | ||
|- | |- | ||
| DNA (clean digest) || | | DNA (clean digest) || 11.0 (~200 ng) | ||
|- | |- | ||
| 10x buffer | | 10x phos. buffer || 1.5 | ||
|- | |- | ||
| phosphatase || 0.5 | | phosphatase || 0.5 | ||
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'''Ligation''' | '''Ligation''' | ||
* Use 20 ng of vector for each ligation = '''1.5 μL pSB1A3''' | * Use 20 ng of vector for each ligation = '''1.5 μL pSB1A3''' | ||
* Use | * Use 3x moles of H2B insert, relative to vector: xμL H2B insert = 3 * 410 bp H2B / 2000 bp pSB1A3 * 20 ng pSB1A3 / 16 ng/μL H2B = at least '''0.8 μL H2B''' | ||
* Use | * Use 3x moles of LOV insert, relative to vector: xμL LOV insert = 3 * 461 bp LOV / 2000 bp pSB1A3 * 20 ng pSB1A3 / 25 ng/μL LOV = at least '''0.5 μL LOV''' | ||
{| class="wikitable" width= | {| class="wikitable" width=700px | ||
| || H2B || LOV || Negative Control | | || H2B || LOV || Negative Control || H2B || LOV || Negative Control | ||
|- | |- | ||
| Insert DNA (2x mol vector) || | | Insert DNA (2x mol vector) || 1.0 μL || 1.0 || --- || 1.0 || 1.0 || --- | ||
|- | |- | ||
| Vector DNA (50 ng) || 1.5 || 1.5 || 1.5 | | Vector DNA (50 ng) || 1.5 || 1.5 || 1.5 || 1.5 || 1.5 || 1.5 | ||
|- | |- | ||
| 2x Roche Rapid Ligation buffer || 5.0 || 5.0 || 5.0 | | 2x Roche Rapid Ligation buffer || 5.0 || 5.0 || 5.0 || 5.0 || 5.0 || 5.0 | ||
|- | |- | ||
| | | T4 ligase || 1.0 NEB || 1.0 NEB || 1.0 NEB || 1.0 Roche || 1.0 Roche || 1.0 Roche | ||
|- | |- | ||
| dH<sub>2</sub>O || | | dH<sub>2</sub>O || 1.5 || 1.5 || 2.5 || 1.5 || 1.5 || 2.5 | ||
|- | |- | ||
| TOTAL || 10.0 || 10.0 || 10.0 | | TOTAL || 10.0 || 10.0 || 10.0 || 10.0 || 10.0 || 10.0 | ||
|- | |- | ||
| colspan="3" | Mix the reaction(s) thoroughly by flicking the tube.<br>Incubate at room temperature for 10 minutes. | | colspan="3" | Mix the reaction(s) thoroughly by flicking the tube.<br>Incubate at room temperature for 10 minutes. |
Revision as of 14:58, 23 November 2012
Project name | <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page <html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html> </html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html> | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
11/21/12Results from yesterday's ligation
PCR program:
Assemblies
> Dephosphorylation (Roche)
Proceed directly to Transformation. See http://openwetware.org/wiki/Haynes:Assembly101
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