Haynes Lab:Notebook/Engineering PC-TFs/2013/02/08 - Revision history

Brady S. Laughlin: /* Summary */

Brady S. Laughlin — Sat, 09 Feb 2013 20:13:30 GMT

Summary

←Older revision		Revision as of 20:13, 9 February 2013
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	<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### -->		<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### -->
-	==~~Summary~~==	+	==2/8/2013==
	* * '''Perform BsmBI/ T4 ligase mediated assembly'''		* * '''Perform BsmBI/ T4 ligase mediated assembly'''
	* BsmBI cuts the DNA fragments and creates complementary overhangs.		* BsmBI cuts the DNA fragments and creates complementary overhangs.
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	\| Total\|\| 10.0 \|\| 10.0		\| Total\|\| 10.0 \|\| 10.0
	\|}		\|}
		+
		+
		+	<font size=3>'''Bacterial transformation'''</font>
		+	* Added total volume (10.0 μL) to 50 μL chemically competent cells (e.g., BL21) in a 2.0 mL tube.
		+	* Incubated on ice for 2 min., heat shock at 42°C for exactly 45 sec., and placed on ice.
		+	* Added 800 μL sterile SOC medium.
		+	* Grow with shaking at 37°C for 30 min.(''Taped tubes to the shaker rack.'')
		+	* Pelleted the cells at top speed in a microcentrifuge for 3 min. at room temperature.
		+	* Discarded the supernatant. Resuspended the cells in 100 μL LB + antibiotic.
		+	* Plate cells on pre-warmed LB agar + antibiotic. (''I plated 100 μL of the cells. Then put beads on plates and shook.) Protocol didn't specify.'') Grow overnight at 37°C.
		+	* Check results tomorrow.
		+
		+	[[Image:plates_1_8_2013.jpg\|400px\|\|left\|]]
		+
		+	*''Still nothing. I'm going to start everything over and see if I can troubleshoot. Vi should be working on Type IIS Assembly this week. So I should have something going by the end of next week.''

Brady S. Laughlin: /* Summary */

Brady S. Laughlin — Sat, 09 Feb 2013 20:11:16 GMT

Summary

←Older revision		Revision as of 20:11, 9 February 2013
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	<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### -->		<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### -->
	==Summary==		==Summary==
-	*	+	* * '''Perform BsmBI/ T4 ligase mediated assembly'''
		+	* BsmBI cuts the DNA fragments and creates complementary overhangs.
		+	* Complementary sticky ends anneal via base pairing.
		+	* T4 ligase seals gaps in the phosphodiester DNA backbone.
		+	{\| {{table}}
		+	\| bgcolor="grey" \| Reagent
		+	\| bgcolor="grey" \| Vol.
		+	\| rowspan=7 \| '''Thermal cycling'''
		+	* [45°C, 2 min.; 16°C 5 min.] x25
		+	* 60°C, 10 min.
		+	* 80°C, 20 min.
		+	* 4°C, ∞
		+	\|-
		+	\| 20 fmol (1 μL) of each DNA part \|\| up to 8.0
		+	\|-
		+	\| 10x T4 ligase buffer (Promega) \|\| 1.0
		+	\|-
		+	\| T4 ligase (NEB) \|\| 0.25
		+	\|-
		+	\| BsmBI \|\| 0.5
		+	\|-
		+	\| dH<sub>2</sub>O \|\| 0.25
		+	\|-
		+	\|   \|\| 10.0 μL
		+	\|}
		+	{\| {{table}} border="1" cellspacing="3" <!-- Golden Gate Rxn. table -->
		+	\|- valign="top"
		+	\| bgcolor="grey" \| Reagent
		+	\| bgcolor="grey" \| 1
		+	\| bgcolor="grey" \| 2
		+	\| bgcolor="grey" \| 3
		+	\|-
		+	\| gg2 pSB1A3 \|\| 1.0 \|\| 1.0 \|\| 1.0
		+	\|-
		+	\| gg3 hPCD \|\| 1.0 \|\| 1.0 \|\| 1.0
		+	\|-
		+	\| BL02 \|\| 1.0 \|\| --- \|\| ---
		+	\|-
		+	\| BL03 \|\| --- \|\| 1.0 \|\| ---
		+	\|-
		+	\| BL04 \|\| --- \|\| --- \|\| 1.0
		+	\|-
		+	\| 10x ligase buffer \|\| 1.0 \|\| 1.0 \|\| 1.0
		+	\|-
		+	\| NEB T4 lgase \|\| 0.25 \|\| 0.25 \|\| 1.0
		+	\|-
		+	\| BsmBI \|\| 0.5 \|\| 0.5 \|\| 0.5
		+	\|-
		+	\| dH<sub>2</sub>O \|\| 5.25 \|\| 5.25 \|\| 5.25
		+	\|-
		+	\| Total\|\| 10.0 \|\| 10.0
		+	\|}
		+

Brady S. Laughlin: Autocreate 2013/02/08 Entry for Haynes_Lab:Notebook/Engineering_PC-TFs

Brady S. Laughlin — Sat, 09 Feb 2013 20:10:03 GMT

Autocreate 2013/02/08 Entry for Haynes_Lab:Notebook/Engineering_PC-TFs

New page

{|{{table}} width="800"
|-
|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Engineering PC-TFs</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>      </html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}}
|-
| colspan="2"|

==Summary==
*


|}

__NOTOC__