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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| dH<sub>2</sub>O || 10.0 || 10.0 | | dH<sub>2</sub>O || 10.0 || 10.0 | ||
|- | |- | ||
| Total || | | Total || 25.0 || 25.0 | ||
|} | |} | ||
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* 4°C ∞ | * 4°C ∞ | ||
{| class="wikitable" border=0 | * Clean up PCR products using the Zymo Clean and Concentrator kit | ||
* Be sure to elute using 15 μL dH<sub>2</sub>O | |||
---- | |||
*'''[[User:Karmella Haynes|---Karmella]] 15:56, 21 November 2012 (EST)''': Digest, purification, and ligation | |||
'''Assemblies''' | |||
# 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: | |||
** In a 30 μL reaction, cut H2B with XbaI and SpeI | |||
** In a 30 μL reaction, cut LOV with XbaI and SpeI | |||
{| class="wikitable" border=1 cellpadding="5" cellspacing="0" | |||
|- | |||
| Reagent || H2B PCR || LOV PCR | |||
|- | |- | ||
| < | | DNA || 15.0 || 15.0 | ||
| | |- | ||
| XbaI || 1.0|| 1.0 | |||
|- | |||
| SpeI || 1.0 || 1.0 | |||
|- | |||
| 10x buffer || 3.0 || 3.0 | |||
|- | |||
| dH<sub>2</sub>O || 10.0 || 10.0 | |||
|- | |||
| Total vol. || 30. 0 || 30.0 | |||
|} | |} | ||
[[Image: | * Run entire 30 μL on a 1% gel (use the big fat-tooth well comb) | ||
* Cut band out of gel (use UV box) | |||
Results from gel recovery: | |||
{| class="wikitable" border=1 cellpadding="5" cellspacing="0" | |||
|- | |||
| Sample|| 260|| 260/280 || ng/μL | |||
|- | |||
| H2B - X/S 1 || 0.016 ||1.807|| 15.961 | |||
|- | |||
| 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_11-21-12.png|Results of from the digests are shown.]] | |||
[[Image:KAH_Fermentas_GeneRuler_1kbplus.jpg]] | [[Image:KAH_Fermentas_GeneRuler_1kbplus.jpg]] | ||
# H2B = 410 | |||
# 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''' (11/23/12) | |||
> Dephosphorylation (Roche) | |||
# pSB1A3 - X/S = 2000 bp | |||
{| class="wikitable" border="0" cellspacing="3" <!-- Dephos table --> | |||
|- | |||
| <u>Reagent</u> || <u>Volume</u> | |||
|- | |||
| DNA (clean digest) || 11.0 (~200 ng) | |||
|- | |||
| 10x phos. buffer || 1.5 | |||
|- | |||
| phosphatase || 0.5 | |||
|- | |||
| dH<sub>2</sub>O || 2.0 | |||
|- | |||
| || 15 μL | |||
|} | |||
* Incubate at 37°C/ 10 min. | |||
* Heat inactivate at 75°C/ 2 min. | |||
* [final] = 200 ng/μL / 15 μL = ~13 ng/μL | |||
'''Ligation''' | |||
* Use 20 ng of vector for each ligation = '''1.5 μL pSB1A3''' | |||
* 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 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=700px | |||
| || H2B || LOV || Negative Control || H2B || LOV || Negative Control | |||
|- | |||
| Insert DNA (2x mol vector) || 1.0 μL || 1.0 || --- || 1.0 || 1.0 || --- | |||
|- | |||
| 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 || 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 || 1.5 || 1.5 || 2.5 || 1.5 || 1.5 || 2.5 | |||
|- | |||
| 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. | |||
|} | |||
Proceed directly to Transformation. See http://openwetware.org/wiki/Haynes:Assembly101 | |||
Revision as of 14:58, 23 November 2012
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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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