User:Keyun Wang/Notebook/Experimental Biological Chemistry I/2012/10/16 - Revision history

Keyun Wang at 02:28, 27 October 2012

2012-10-27T02:28:04Z

←Older revision		Revision as of 02:28, 27 October 2012
Line 21:		Line 21:
	Ext. Coefficient: 534,6000 L/(mole·cm)		Ext. Coefficient: 534,6000 L/(mole·cm)
	Amount of moles=34.4moles		Amount of moles=34.4moles
		+
	* The concentration of primer E34K reverse was calculated, and amount of diluted primer needed for PCR was determined the following way:		* The concentration of primer E34K reverse was calculated, and amount of diluted primer needed for PCR was determined the following way:
	Target concentration: 100ng/uL		Target concentration: 100ng/uL
Line 29:		Line 30:
	**172.41μL of 3.4×10<sup>4</sup>M reverse primer was pipetted into a 1.5mL microcentrifuge tube, and 827.58μL of sterile water was added to make up a total of 1mL 100ng/μL reverse primer.		**172.41μL of 3.4×10<sup>4</sup>M reverse primer was pipetted into a 1.5mL microcentrifuge tube, and 827.58μL of sterile water was added to make up a total of 1mL 100ng/μL reverse primer.
	**100ng/μL reverse primer was made at 1mL.		**100ng/μL reverse primer was made at 1mL.
		+
	* With 100ng/μL of forward primer of E34K made, PCR solution was made with the following reactants:		* With 100ng/μL of forward primer of E34K made, PCR solution was made with the following reactants:
	1μL of forward E34K primer		1μL of forward E34K primer
Line 39:		Line 41:
	**Turbo DNA polymerase was added last and right before PCR solution was placed on Thermocycler		**Turbo DNA polymerase was added last and right before PCR solution was placed on Thermocycler
	**Two of PCR solutions were made		**Two of PCR solutions were made
		+
	*The Thermocycler was programed in the following way to detach and reanneal DNA to allow DNA amplification:		*The Thermocycler was programed in the following way to detach and reanneal DNA to allow DNA amplification:
	Heating for 2minutes at 95°C		Heating for 2minutes at 95°C
Line 47:		Line 50:
	Maintaining temperature at 72°C		Maintaining temperature at 72°C
	Cooling back to 0°C for plasma stability		Cooling back to 0°C for plasma stability
		+
	* New sets of Au/BSA solutions was made with the following ratios:		* New sets of Au/BSA solutions was made with the following ratios:
	60, 80, 100, 120, 128, 130, 132, 133, 134, 136, 138, 140, 160, 170		60, 80, 100, 120, 128, 130, 132, 133, 134, 136, 138, 140, 160, 170
Line 54:		Line 58:
	0.0398g BSA ÷ 66,000g/mol = 3.01×10<sup>-5</sup>M = 30.1μM BSA		0.0398g BSA ÷ 66,000g/mol = 3.01×10<sup>-5</sup>M = 30.1μM BSA
	**Stock for HAuCl4 from [[User:Keyun Wang/Notebook/Experimental Biological Chemistry I/2012/10/03\|10/03/12]] was used due to long shelf life		**Stock for HAuCl4 from [[User:Keyun Wang/Notebook/Experimental Biological Chemistry I/2012/10/03\|10/03/12]] was used due to long shelf life
-	* The amount of HAuCl4, BSA, and water was calculated via excel to make Au/BSA solutions with above ratios, show in table below:

		+	* The amount of HAuCl4, BSA, and water was calculated via excel to make Au/BSA solutions with above ratios, show in table below:
		+	{\| {{table}}
		+	\| align="center" style="background:#f0f0f0;"\|'''Au/BSA ratio'''
		+	\| align="center" style="background:#f0f0f0;"\|'''Mol of BSA added'''
		+	\| align="center" style="background:#f0f0f0;"\|'''Volume Luminol Added[uL]'''
		+	\| align="center" style="background:#f0f0f0;"\|'''Volume BSA added[uL]'''
		+	\| align="center" style="background:#f0f0f0;"\|'''Volume H2O Added[uL]'''
		+	\|-
		+	\| 60\|\|3.44E-08\|\|80\|\|2310.51\|\|7609.49
		+	\|-
		+	\| 80\|\|2.57E-08\|\|80\|\|1728.86\|\|8191.14
		+	\|-
		+	\| 100\|\|2.05E-08\|\|80\|\|1379.87\|\|8540.13
		+	\|-
		+	\| 120\|\|1.71E-08\|\|80\|\|1147.2\|\|8772.8
		+	\|-
		+	\| 128\|\|1.60E-08\|\|80\|\|1074.5\|\|8845.5
		+	\|-
		+	\| 130\|\|1.58E-08\|\|80\|\|1057.72\|\|8862.28
		+	\|-
		+	\| 132\|\|1.55E-08\|\|80\|\|1041.45\|\|8878.55
		+	\|-
		+	\| 133\|\|1.54E-08\|\|80\|\|1033.5\|\|8886.5
		+	\|-
		+	\| 134\|\|1.53E-08\|\|80\|\|1025.66\|\|8894.34
		+	\|-
		+	\| 136\|\|1.51E-08\|\|80\|\|1010.34\|\|8909.66
		+	\|-
		+	\| 138\|\|1.48E-08\|\|80\|\|995.47\|\|8924.53
		+	\|-
		+	\| 140\|\|1.46E-08\|\|80\|\|981.02\|\|8938.98
		+	\|-
		+	\| 160\|\|1.28E-08\|\|80\|\|856.38\|\|9063.62
		+	\|-
		+	\| 170\|\|1.20E-08\|\|80\|\|805.05\|\|9114.95
		+	\|}
		+	** The solutions with above volume of different substrate were mixed with separate in glass test tubes, wrapped in aluminum foil, and placed in incubator at 85°C for 4 hours
	<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### -->		<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### -->
	\|}		\|}

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Keyun Wang at 22:54, 26 October 2012

2012-10-26T22:54:04Z

←Older revision		Revision as of 22:54, 26 October 2012
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	==Procedure==		==Procedure==
-		+	* Concentration needed to dilute the primer E34K reverse was calculated with the below information:
-		+	Melting Temperature in 50mM NaCl=62.2°C
		+	Molecular Weight=16,877.0g/mol
		+	Amount of miligrams present=0.58mg
		+	Strand: 5'-ATG TTA AATCTA AAT GGC AAT GTA ATT TAA CTT TGG GAA TTC TTC TTC ATA TTT C-3'
		+	GC content=27.2%
		+	nmoles/OD260=1.9
		+	ug/OD260=31.6
		+	Ext. Coefficient: 534,6000 L/(mole·cm)
		+	Amount of moles=34.4moles
		+	* The concentration of primer E34K reverse was calculated, and amount of diluted primer needed for PCR was determined the following way:
		+	Target concentration: 100ng/uL
		+	0.58mg=0.58·10<sup>6</sup>ng
		+	0.58·10<sup>6</sup>ng / 1000μL H<sub>2</sub>O = 580ng/μL
		+	100ng/uL x 1000uL / 580ng/uL = 172.41μL
		+	**1mL of sterile water was added into reverse primer, making the concentration of the reverse primer 3.4×10<sup>4</sup>M.
		+	**172.41μL of 3.4×10<sup>4</sup>M reverse primer was pipetted into a 1.5mL microcentrifuge tube, and 827.58μL of sterile water was added to make up a total of 1mL 100ng/μL reverse primer.
		+	**100ng/μL reverse primer was made at 1mL.
		+	* With 100ng/μL of forward primer of E34K made, PCR solution was made with the following reactants:
		+	1μL of forward E34K primer
		+	1μL of reverse E34K primer
		+	1μL of ADA wild strand
		+	0.4μL of 25mM dNTPs
		+	5μL of 10x cloned Pfu buffer
		+	40.6μL of sterile water
		+	1μL of 2.5μL Turbo DNA polymerase
		+	**Turbo DNA polymerase was added last and right before PCR solution was placed on Thermocycler
		+	**Two of PCR solutions were made
		+	*The Thermocycler was programed in the following way to detach and reanneal DNA to allow DNA amplification:
		+	Heating for 2minutes at 95°C
		+	30 times of the following procedure:
		+	Heating for 30 seconds at 95°C
		+	Cooling for 30 seconds at -5°C
		+	Reheating back for 1 minute at 72°C
		+	Maintaining temperature at 72°C
		+	Cooling back to 0°C for plasma stability
		+	* New sets of Au/BSA solutions was made with the following ratios:
		+	60, 80, 100, 120, 128, 130, 132, 133, 134, 136, 138, 140, 160, 170
		+	**Stock solution of BSA at 3μM and HAuCl4 at 26mM was made the following way:
		+	Molecular weight of BSA: 66,000g/mol
		+	0.0398g of BSA in powder form was weighted out and diluted in 20mL of water
		+	0.0398g BSA ÷ 66,000g/mol = 3.01×10<sup>-5</sup>M = 30.1μM BSA
		+	**Stock for HAuCl4 from [[User:Keyun Wang/Notebook/Experimental Biological Chemistry I/2012/10/03\|10/03/12]] was used due to long shelf life
		+	* The amount of HAuCl4, BSA, and water was calculated via excel to make Au/BSA solutions with above ratios, show in table below:

	<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### -->		<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### -->

Keyun Wang at 21:23, 26 October 2012

2012-10-26T21:23:11Z

←Older revision		Revision as of 21:23, 26 October 2012
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	{\|{{table}} width="800"		{\|{{table}} width="800"
	\|-		\|-
-	\|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;"> Experimental Biological Chemistry I</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>}}		\|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"\|		\| colspan="2"\|
	<!-- ##### 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. ##### -->
-	==~~Entry title~~==	+	==Purpose==
-	* ~~Insert content here..~~.	+	* Perform polymerase chain reaction to design ADA plasmids with mutation at the 34th amino acid position from glutamate to lysine: E34K.
		+	* Make new Au/BSA solutions for more fibers in order to perform Atomic Absorption spectroscopy to find out gold concentration in solution
		+
		+	==Procedure==
		+

Keyun Wang: Autocreate 2012/10/16 Entry for User:Keyun_Wang/Notebook/Experimental_Biological_Chemistry_I

2012-10-26T21:15:01Z

Autocreate 2012/10/16 Entry for User:Keyun_Wang/Notebook/Experimental_Biological_Chemistry_I

New page

{|{{table}} width="800"
|-
|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>      </html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}}
|-
| colspan="2"|

==Entry title==
* Insert content here...


|}

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