Luckau Protocols:1Kb Ladder - Revision history

Tara K. Luckau at 19:51, 20 December 2012

Tara K. Luckau — Thu, 20 Dec 2012 19:51:22 GMT

←Older revision		Revision as of 19:51, 20 December 2012
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	* STK at 1 µg/µL		* STK at 1 µg/µL
	* [[Media:Invitrogen_1kb_plus.pdf]]		* [[Media:Invitrogen_1kb_plus.pdf]]
-		+	:<small>(We used to use Invitrogen's 1Kb DNA Ladder, catalog# 15615-016, [[Media:Invitrogen_1KbLadderProductManual.pdf]], [[Media:1kb Gel Ladder Map.jpg]], but they have since discontinued this product. The two products are compatible.)</small>
-		+
-	::(We used to use Invitrogen's 1Kb DNA Ladder, catalog# 15615-016, [[Media:Invitrogen_1KbLadderProductManual.pdf]], [[Media:1kb Gel Ladder Map.jpg]], but they have since discontinued this product. The two products are compatible.)	+

Tara K. Luckau at 19:48, 20 December 2012

Tara K. Luckau — Thu, 20 Dec 2012 19:48:14 GMT

←Older revision		Revision as of 19:48, 20 December 2012
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	==Ladder Map==		==Ladder Map==
-	: [[Image:Invitrogen1Kbv1KbPlusLadder.jpg]]	+	: [[Image:Invitrogen1Kbv1KbPlusLadder.jpg\|300 px]]

Tara K. Luckau at 22:11, 24 October 2012

Tara K. Luckau — Wed, 24 Oct 2012 22:11:07 GMT

←Older revision		Revision as of 22:11, 24 October 2012
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-	::(We used to use Invitrogen's 1Kb DNA Ladder, catalog# 15615-016, [[Media:Invitrogen_1KbLadderProductManual.pdf]], [[Media:1kb Gel Ladder Map.jpg~~\|300 px~~]], but they have since discontinued this product. The two products are compatible.)	+	::(We used to use Invitrogen's 1Kb DNA Ladder, catalog# 15615-016, [[Media:Invitrogen_1KbLadderProductManual.pdf]], [[Media:1kb Gel Ladder Map.jpg]], but they have since discontinued this product. The two products are compatible.)

Tara K. Luckau at 22:09, 24 October 2012

Tara K. Luckau — Wed, 24 Oct 2012 22:09:50 GMT

←Older revision		Revision as of 22:09, 24 October 2012
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-	:::(We used to use Invitrogen's 1Kb DNA Ladder, catalog# 15615-016, [[Media:Invitrogen_1KbLadderProductManual.pdf]], but they have since discontinued this product. The two products are compatible.)	+	::(We used to use Invitrogen's 1Kb DNA Ladder, catalog# 15615-016, [[Media:Invitrogen_1KbLadderProductManual.pdf]], [[Media:1kb Gel Ladder Map.jpg\|300 px]], but they have since discontinued this product. The two products are compatible.)


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	===Other Knowns===		===Other Knowns===
	* well width (21-tooth comb) = 7mm		* well width (21-tooth comb) = 7mm
		+	:: calculations made for 6mm width, since they end up being more convenient


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	===Calculations===		===Calculations===
-	* ~~7mm~~ x (0.1µg ladder)/(1mm width) x (1µL/1µg) = 0.~~7µL~~ ladder per well	+	* 6mm x (0.1µg ladder)/(1mm width) x (1µL/1µg) = 0.6µL ladder per well
-	* <math>7 \mbox{ mm} \bullet \tfrac{0.1 \mbox{ } \mu \mbox{g ladder}}{1 \mbox{ mm width}}\ \bullet \tfrac{1 \mbox{ } \mu \mbox{L}}{1 \mbox{ } \mu \mbox{g}}\ = 0.7 \mbox{ } \mu \mbox{L ladder per well}</math>	+	* <math>6 \mbox{ mm} \bullet \tfrac{0.1 \mbox{ } \mu \mbox{g ladder}}{1 \mbox{ mm width}}\ \bullet \tfrac{1 \mbox{ } \mu \mbox{L}}{1 \mbox{ } \mu \mbox{g}}\ = 0.6 \mbox{ } \mu \mbox{L ladder per well}</math>
	* Per well: 0.6 µL ladder + 4.4 µL H<sub>2</sub>O + 1 µL loading dye = 6 µL ladder mix		* Per well: 0.6 µL ladder + 4.4 µL H<sub>2</sub>O + 1 µL loading dye = 6 µL ladder mix

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-	==~~Migration of gel load dye &~~ Ladder Map==	+	==Ladder Map==
		+	: [[Image:Invitrogen1Kbv1KbPlusLadder.jpg]]
		+
		+
		+	==Migration of Gel Load Dyes (using old ladder)==
	* Bromophenol Blue (175bp) and Xylene Cyanol FF (1500bp)		* Bromophenol Blue (175bp) and Xylene Cyanol FF (1500bp)
-	: [[Image:AgaroseGel_2_XCBPB~~.jpg]] ------------- [[Image:1kb Gel Ladder Map~~.jpg\|~~300~~ px]]	+	: [[Image:AgaroseGel_2_XCBPB.jpg\|100 px]]

Tara K. Luckau at 18:45, 24 October 2012

Tara K. Luckau — Wed, 24 Oct 2012 18:45:13 GMT

←Older revision		Revision as of 18:45, 24 October 2012
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	==Purpose==		==Purpose==

-	Nucleic acid ladder is used as a size standard in agarose gel electrophoresis. It contains DNA molecules of known lengths. The ladder is pre-mixed with gel load buffer (color dyes with varying mobility on an agarose gel) for direct ~~addition of PCR product (or genomic DNA) and~~ transfer to the gel.	+	Nucleic acid ladder is used as a size standard in agarose gel electrophoresis. It contains DNA molecules of known lengths. The ladder is pre-mixed with gel load buffer (color dyes with varying mobility on an agarose gel) for direct transfer to the gel.


	==Materials==		==Materials==

-	===Invitrogen 1Kb Ladder===	+	===Invitrogen 1Kb Plus DNA Ladder===
-	* catalog# ~~15615~~-~~016~~	+	* catalog# 10787-018
-	* ~~Invitrogen says to~~ use: 0.1µg ladder / mm width of well	+	* recommended use: 0.1µg ladder / mm width of well
-	* STK at 1~~.0µg~~/µL	+	* STK at 1 µg/µL
-	* [[Media:Invitrogen_1KbLadderProductManual.pdf]]	+	* [[Media:Invitrogen_1kb_plus.pdf]]
		+
		+
		+	:::(We used to use Invitrogen's 1Kb DNA Ladder, catalog# 15615-016, [[Media:Invitrogen_1KbLadderProductManual.pdf]], but they have since discontinued this product. The two products are compatible.)
		+

	===Promega Blue/Orange Loading Dye, 6x===		===Promega Blue/Orange Loading Dye, 6x===
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	===Other Knowns===		===Other Knowns===
-	* well width (24-tooth comb) = ~~6mm~~	+	* well width (21-tooth comb) = 7mm


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	===Calculations===		===Calculations===
-	* ~~6mm~~ x (0.1µg ladder)/(1mm width) x (1µL/1µg) = 0.~~6µL~~ ladder per well	+	* 7mm x (0.1µg ladder)/(1mm width) x (1µL/1µg) = 0.7µL ladder per well
-	* Per well: 0.~~6µL~~ ladder + 4.~~4µL H2O~~ + ~~1µL~~ loading dye = ~~6µL total~~ ladder mix	+	* <math>7 \mbox{ mm} \bullet \tfrac{0.1 \mbox{ } \mu \mbox{g ladder}}{1 \mbox{ mm width}}\ \bullet \tfrac{1 \mbox{ } \mu \mbox{L}}{1 \mbox{ } \mu \mbox{g}}\ = 0.7 \mbox{ } \mu \mbox{L ladder per well}</math>
		+	* Per well: 0.6 µL ladder + 4.4 µL H<sub>2</sub>O + 1 µL loading dye = 6 µL ladder mix

	===Make Ladder Mix===		===Make Ladder Mix===
-	~~120µL~~ ladder + ~~880µL H2O~~ + ~~200µL~~ loading dye = ~~1200µL~~ ladder mix	+	120 µL ladder + 880 µL H<sub>2</sub>O + 200 µL loading dye = 1200 µL ladder mix

Tara K. Luckau: /* Promega Blue/Orange Loading Dye, 6x */

Tara K. Luckau — Mon, 31 Oct 2011 19:49:42 GMT

Promega Blue/Orange Loading Dye, 6x

←Older revision		Revision as of 19:49, 31 October 2011
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	* catalog# G1881		* catalog# G1881
	* use at 1x concentration		* use at 1x concentration
		+	* contains:
		+	:: 0.4% orange G
		+	:: 0.03% bromophenol blue
		+	:: 0.03% xylene cyanol FF
		+	:: 15% Ficoll® 400 (for density; replaces glycerol)
		+	:: 10mM Tris-HCl (pH 7.5)
		+	:: 50mM EDTA (pH 8.0)
		+	* In a 0.5–1.4% agarose gel in 0.5X TBE:
		+	:: xylene cyanol FF migrates at approximately 4kb
		+	:: bromophenol blue at approximately 300bp
		+	:: orange G at approximately 50bp.

	===Other Knowns===		===Other Knowns===

Tara K. Luckau at 22:55, 28 October 2011

Tara K. Luckau — Fri, 28 Oct 2011 22:55:35 GMT

←Older revision		Revision as of 22:55, 28 October 2011
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	[[Luckau_Protocols \| Tara's Protocols]]		[[Luckau_Protocols \| Tara's Protocols]]
	\|}		\|}
		+

	==Purpose==		==Purpose==
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	===Make Ladder Mix===		===Make Ladder Mix===
	120µL ladder + 880µL H2O + 200µL loading dye = 1200µL ladder mix		120µL ladder + 880µL H2O + 200µL loading dye = 1200µL ladder mix
		+
		+
		+	==Migration of gel load dye & Ladder Map==
		+	* Bromophenol Blue (175bp) and Xylene Cyanol FF (1500bp)
		+	: [[Image:AgaroseGel_2_XCBPB.jpg]] ------------- [[Image:1kb Gel Ladder Map.jpg\|300 px]]

Tara K. Luckau at 03:50, 3 May 2011

Tara K. Luckau — Tue, 03 May 2011 03:50:56 GMT

←Older revision		Revision as of 03:50, 3 May 2011
Line 1:		Line 1:
-	Ladder Mix	+	{\|{{table}} width="900"
		+	\|-
		+	\| width="600" style="background-color: #BCED91;" align="center"\| <span style="font-size:32px;"> 1Kb Ladder Mix </span>

-	[~~edit~~]~~Manufacturer specifications~~	+	\|align="left" style="background-color: #9DB68C;" \|
-	0.1µg ladder / mm width of well	+	<span style="font-size:16px;"> [[User:Tara_K._Luckau \| Tara K. Luckau's Home Page]] </span>
-	STK at 1.0µg/µL	+
-	Media:Invitrogen_1KbLadderProductManual.pdf	+	[[User:Tara_K._Luckau/Notebook/Team_ConGen \| Conservation Genetics Lab Notebook]]
-	~~[edit~~]Other Knowns	+
-	well width (24-tooth comb) = 6mm	+	[[Luckau_Protocols \| Tara's Protocols]]
-	~~Jeremy (Bohonak lab) uses 5µL PCR product + 1.5µL dye~~	+	\|}
-	~~[edit]~~Calculations	+
-	6mm x (0.1µg ladder)/(1mm width) x (1µL/1µg) = 0.6µL ladder per well	+	==Purpose==
-	Per well: 0.6µL ladder + 4.4µL H2O + ~~1.5µL~~ loading dye = ~~6.5µL~~ total ladder mix	+
-	~~(loading dye~~ = ~~xylene cyanol and bromophenol, made by Bohonak lab)~~	+	Nucleic acid ladder is used as a size standard in agarose gel electrophoresis. It contains DNA molecules of known lengths. The ladder is pre-mixed with gel load buffer (color dyes with varying mobility on an agarose gel) for direct addition of PCR product (or genomic DNA) and transfer to the gel.
-	~~[edit]~~Make Ladder Mix	+
-	120µL ladder + 880µL H2O + ~~300µL~~ loading dye = ~~1300µL~~ ladder mix	+
		+	==Materials==
		+
		+	===Invitrogen 1Kb Ladder===
		+	* catalog# 15615-016
		+	* Invitrogen says to use: 0.1µg ladder / mm width of well
		+	* STK at 1.0µg/µL
		+	* [[Media:Invitrogen_1KbLadderProductManual.pdf]]
		+
		+	===Promega Blue/Orange Loading Dye, 6x===
		+	* catalog# G1881
		+	* use at 1x concentration
		+
		+	===Other Knowns===
		+	* well width (24-tooth comb) = 6mm
		+
		+
		+	==Protocol==
		+
		+	===Calculations===
		+	* 6mm x (0.1µg ladder)/(1mm width) x (1µL/1µg) = 0.6µL ladder per well
		+	* Per well: 0.6µL ladder + 4.4µL H2O + 1µL loading dye = 6µL total ladder mix
		+
		+	===Make Ladder Mix===
		+	120µL ladder + 880µL H2O + 200µL loading dye = 1200µL ladder mix

Tara K. Luckau: New page: Ladder Mix [edit]Manufacturer specifications 0.1µg ladder / mm width of well STK at 1.0µg/µL Media:Invitrogen_1KbLadderProductManual.pdf [edit]Other Knowns well width (24-tooth comb) =...

Tara K. Luckau — Mon, 02 May 2011 22:43:52 GMT

New page: Ladder Mix [edit]Manufacturer specifications 0.1µg ladder / mm width of well STK at 1.0µg/µL Media:Invitrogen_1KbLadderProductManual.pdf [edit]Other Knowns well width (24-tooth comb) =...

New page

Ladder Mix

[edit]Manufacturer specifications
0.1µg ladder / mm width of well
STK at 1.0µg/µL
Media:Invitrogen_1KbLadderProductManual.pdf
[edit]Other Knowns
well width (24-tooth comb) = 6mm
Jeremy (Bohonak lab) uses 5µL PCR product + 1.5µL dye
[edit]Calculations
6mm x (0.1µg ladder)/(1mm width) x (1µL/1µg) = 0.6µL ladder per well
Per well: 0.6µL ladder + 4.4µL H2O + 1.5µL loading dye = 6.5µL total ladder mix
(loading dye = xylene cyanol and bromophenol, made by Bohonak lab)
[edit]Make Ladder Mix
120µL ladder + 880µL H2O + 300µL loading dye = 1300µL ladder mix