Haynes Lab:Notebook/Investigating Photo-Switchable Synthetic Nucleosomes/2012/12/05: Difference between revisions

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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Project name</span>
|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Project name</span>
|style="background-color: #F2F2F2" align="center"|<html><img src="/images/9/94/Report.png" border="0" /></html> [[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>[[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}}
|style="background-color: #F2F2F2" align="center"|[[File:Report.png|frameless|link={{#sub:{{FULLPAGENAME}}|0|-11}}]][[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|[[File:Resultset_previous.png|frameless|link={{#lnpreventry:{{FULLPAGENAME}}}}]][[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]][[File:Resultset_next.png|frameless|link={{#lnnextentry:{{FULLPAGENAME}}}}]]}}
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==December 5, 2012==
==December 5, 2012==
==11/21/12==
'''Results from last 11/23/12 ligation'''
 
'''Results from last week's ligation'''
* None of the colonies on any plates grew
* None of the colonies on any plates grew
* Will retry cloning for each (H2B, LOV) by using the Gibson assembly method
* Will retry cloning for each (H2B, LOV) by using the Gibson assembly method
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* Note: Primers add a XbaI site upstream and the SpeI site downstream
* Note: Primers add a XbaI site upstream and the SpeI site downstream
* Final Gibson assembly products
* Final Gibson assembly products
#pSB1A3 left end / H2B / LOV +His tag/ pSB1A3 right end
# pSB1A3 left end / H2B / LOV +His tag/ pSB1A3 right end
#pSB1A3 left end / LOV / H2B +His tag/ pSB1A3 right end
# pSB1A3 left end / LOV / H2B +His tag/ pSB1A3 right end


*For H2B-LOV construct
*For H2B-LOV construct
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* Clean up PCR products using the Zymo Clean and Concentrator kit
* Clean up PCR products using the Zymo Clean and Concentrator kit
* Be sure to elute using 15 μL dH<sub>2</sub>O
* 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"
{| class="wikitable" border=1 cellpadding="5" cellspacing="0"
|-
|-
| Reagent || H2B PCR || LOV PCR
|Construct || Product || 260 || 280 || 260/280 || ng/μL
|-
|-
| DNA || 15.0 || 15.0
|H2B-LOV || H2B 1 || 0.061 || 0.033 || 1.863 || 60.545
|-
|-
| XbaI || 1.0|| 1.0
|H2B-LOV || H2B 2 || 0.056 || 0.03 || 1.857 || 56.22
|-
|-
| SpeI || 1.0 || 1.0
|H2B-LOV || LOV 1 || 0.063 || 0.033 || 1.903 || 62.673
|-
|-
| 10x buffer || 3.0 || 3.0
|H2B-LOV || LOV 2 || 0.06 || 0.04 || 1.85 || 59.844
|-
|-
| dH<sub>2</sub>O || 10.0 || 10.0
|LOV-H2B || H2B 1 || 0.072 || 0.033 || 1.807 || 72.381
|-
|-
| Total vol. || 30. 0 || 30.0
|LOV-H2B || H2B 2 || 0.06 || 0.036 || 1.799 || 59.925
|}
 
 
* 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-H2B || LOV 1 || 0.006 || 0.036 || 1.849 || 65.881
|-
|-
| LOV - X/S 2 || 0.025|| 1.883 || 25.363
|LOV-H2B || LOV 2 || 0.065 || 0.035 || 1.839 || 65.285
|}
|}
[[Image:VN_Gel_11-21-12.png|Results of from the digests are shown.]]
[[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
|-
| &nbsp; || 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
| &nbsp; || 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


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Latest revision as of 22:19, 26 September 2017

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December 5, 2012

Results from last 11/23/12 ligation

  • None of the colonies on any plates grew
  • Will retry cloning for each (H2B, LOV) by using the Gibson assembly method
  • Repeat PCR of H2B and LOV inserts with new primers
  • Note: Primers add a XbaI site upstream and the SpeI site downstream
  • Final Gibson assembly products
  1. pSB1A3 left end / H2B / LOV +His tag/ pSB1A3 right end
  2. pSB1A3 left end / LOV / H2B +His tag/ pSB1A3 right end
  • For H2B-LOV construct
  1. H2B-GFP plasmid + 1A3/H2B fwd + H2B/LOV rev
  2. LOV plasmid + H2B/LOV fwd + LOV+his/1A3 rev
  • For LOV-H2B construct
  1. H2B-GFP plasmid + 1A3/LOV fwd + LOV/H2B rev
  2. LOV plasmid + LOV/H2B fwd + H2B+his/1A3 rev

Performed table reactions twice: once for those used in H2B-LOV construct, once for those used in LOV-H2B construct

Reagents H2B LOV
Plasmid DNA 0.5 μL 0.5
primer 1 (10 μM) 1.0 1.0
primer 2 (10 μM) 1.0 1.0
2x GoTaq mix 12.5 12.5
dH2O 10.0 10.0
Total 25.0 25.0

PCR program:

  • 95°C, 3 min.
  • [95°C, 30 sec; 57°C, 30 sec.; 72°C, 30 sec.] x35 cycles
  • 72°C, 3 min.
  • 4°C ∞
  • Clean up PCR products using the Zymo Clean and Concentrator kit
  • Be sure to elute using 15 μL dH2O
Construct Product 260 280 260/280 ng/μL
H2B-LOV H2B 1 0.061 0.033 1.863 60.545
H2B-LOV H2B 2 0.056 0.03 1.857 56.22
H2B-LOV LOV 1 0.063 0.033 1.903 62.673
H2B-LOV LOV 2 0.06 0.04 1.85 59.844
LOV-H2B H2B 1 0.072 0.033 1.807 72.381
LOV-H2B H2B 2 0.06 0.036 1.799 59.925
LOV-H2B LOV 1 0.006 0.036 1.849 65.881
LOV-H2B LOV 2 0.065 0.035 1.839 65.285


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