Catherinel Notebook1 - Revision history

Cadoyle: /* cadoyle, 23 January 2014 */

2014-01-24T13:42:06Z

‎cadoyle, 23 January 2014

Cadoyle: /* cadoyle, 21 January 2014 */

2014-01-23T17:56:22Z

‎cadoyle, 21 January 2014

Cadoyle: /* cadoyle, 17 January 2014 */

2014-01-23T17:55:40Z

‎cadoyle, 17 January 2014

Cadoyle: /* cadoyle, 12 January 2014 */

2014-01-17T18:45:27Z

‎cadoyle, 12 January 2014

Cadoyle: /* cadoyle, 12 January 2014 */

2014-01-17T18:41:49Z

‎cadoyle, 12 January 2014

Cadoyle: /* cadoyle, 13 January 2014 */

2014-01-17T18:40:17Z

‎cadoyle, 13 January 2014

Cadoyle: /* cadoyle, 14 January 2014 */

2014-01-17T18:39:12Z

‎cadoyle, 14 January 2014

Cadoyle: /* cadoyle, 6 December 2013 */

2014-01-17T18:29:39Z

‎cadoyle, 6 December 2013

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Cadoyle: /* cadoyle, 6 December 2013 */

2013-12-06T19:07:58Z

‎cadoyle, 6 December 2013

Cadoyle: /* cadoyle, 5 December 2013 */

2013-12-06T13:46:12Z

‎cadoyle, 5 December 2013

@@ Line 2,629: / Line 2,629: @@
 .5 µL T4 DNA Ligase
 µL final volume
 I added 1 ul of 3.2ng/ul of caffeine and theophylline riboswitches to separate tubes. I also did a control with just plasmid. I added 1ul of water in place of the riboswitch for the control.

@@ Line 2,560: / Line 2,560: @@
 The values look great. Amplification worked well. I will save plasmid for GGA ligation once the G-block riboswitches arrive. My final volume is 18ul.
--22-14
+==[[User:Catherine|cadoyle]], 22 January 2014==
 Riboswitches should arrive today.
 This is plan of action for the next tow weeks. Things are subject to change.
@@ Line 2,609: / Line 2,609: @@
 Tomorrow I will perform a colony PCR to verify if the ligation and transformation were successful.
--23-14
+==[[User:Catherine|cadoyle]], 23 January 2014==
 I check the transformations. The control had three small colonies. Caffeine and theophylline riboswitch both had one very small colony. Either it needs more time to grow or the plates are bad. I will let them grow a little longer. The plates were made in September and the antibiotic resistance might be gone. I can check the plates by just plating some cells on them and see if they grow today. I might need to make more plates.

← Older revision		Revision as of 17:55, 23 January 2014
Line 2,530:		Line 2,530:

	I ran the xanthine riboswitches through the basechecker.py and found none the RBS's or start codons of the riboswitches base paired to the aptamer. Also, earlier I ran the switches through test.py and the difference in free energy of each riboswitch between the ON and OFF state is greater the binding energy of the xanthine aptamer. I emailed Dr. Campbell to let him know.		I ran the xanthine riboswitches through the basechecker.py and found none the RBS's or start codons of the riboswitches base paired to the aptamer. Also, earlier I ran the switches through test.py and the difference in free energy of each riboswitch between the ON and OFF state is greater the binding energy of the xanthine aptamer. I emailed Dr. Campbell to let him know.
		+
		+	==[[User:Catherine\|cadoyle]], 18 January 2014==
		+
		+	Today I finished writing up paragraph summaries for all the biology seminars I attended last semester. Also, I worked on editing my thesis and adding the primers I designed this week to the methods section.
		+
		+	==[[User:Catherine\|cadoyle]], 19-20 January 2014==
		+	I worked on editing my thesis. I finished all the edits from the draft due at the end of last semester. Also, I added what did last week with the primers and riboswitch synthesis designs. The primers arrived today. I stored them the -20C freezer in the lab.
		+	I ran a PCR reaction on J19007 the receiving plasmid with primers J19007 G- Forward and J19007 reverse following this protocol [http://www.bio.davidson.edu/courses/Molbio/Protocols/pcr.html]. The Tm for forward was 67 and for the reverse was 61. I set the annealing temp to 56 and elongation to 3:30min because the final product should 2,984bp. Tomorrow I will run a gel to verify iPCR size.
		+
		+	==[[User:Catherine\|cadoyle]], 21 January 2014==
		+
		+	Gel to verify iPCR product:
		+	I made a 0.4% gel using 0.24g of agarose and 60ml of 0.5X TBE. I added 2ul of Midori green and cooled. I cleaned the gel box and added new 0.5X TBE buffer. In lane 1 I put 5ul of 1kb ladder. Lane 2: 5ul of J100079 Lane 3: 5ul of J10079 iPCR. I ran the gel at 100V for 30 minutes.
		+
		+	[[File:2014-01-21 09hr 51min-4.jpg]]
		+
		+	The control of J100079 is at about 3119bp (lane2) and the iPCR product is little smaller at about 2984bp (lane 3). Amplification and the size of the product looks good. I will clean up the PCR product and save for GGA ligation.
		+
		+	Ethanol Precipitation of iPCR product
		+	Following this protocol I cleaned the iPCR product for GGA ligation [http://www.bio.davidson.edu/courses/Molbio/Protocols/clean_short.html]. I let the product incubate -70C for 20 minutes and resuspended in 20ul of water.
		+
		+	Nanodrop of cleaned iPCR product:
		+	1556.3 ng/ul
		+	260: 31.126
		+	280: 16.949
		+	260/280: 1.84
		+	260/230: 2.34
		+
		+	The values look great. Amplification worked well. I will save plasmid for GGA ligation once the G-block riboswitches arrive. My final volume is 18ul.
		+
		+	1-22-14
		+	Riboswitches should arrive today.
		+	This is plan of action for the next tow weeks. Things are subject to change.
		+	[[File: Screen shot 2014-01-22 at 6.53.34 AM.png]]
		+	[[FIle: Screen shot 2014-01-22 at 6.53.41 AM.png]]
		+
		+	I have a question on the best way to test the constructs. I am thinking liquid but will talk to Dr. C today about it. I will need to check if we have enough plates and media etc.
		+
		+	We have enough plates for a least a couple of weeks.
		+
		+	The riboswitches arrived. I need to re-suspend them in TE to last up to 24 months. If I re-suspend in water they will only last for a month.
		+
		+	I made 10X TE stock:
		+	5ml of 1M Tris, pH 8.0
		+	1ml of 0.5M EDTA
		+	44ml of water
		+
		+	I then diluted the stock to 1X TE
		+	I took 10mls of 10X and 90ml of water
		+
		+	To re-suspend the riboswitches I centrifuged the tube for 3−5 sec at a minimum of 3000 x g to pellet the material to the bottom of the tube. I added 20 μL TE to the tube for a final concentration of 10 ng/μL. Briefly vortex and centrifuged.
		+	GGA of Caffeine and Theophylline riboswitch with J100079 Plasmid
		+
		+	1 µL (50 ng) plasmid containing J100079
		+	5µL dH2O
		+	1 µL 10X Promega Ligase Buffer
		+	1 µL 500 mM KOAc
		+	0.5 µL Bsa I high fidelity (HF) restriction enzyme
		+	0.5 µL T4 DNA Ligase
		+	9 µL final volume
		+
		+	I added 1 ul of 10ng/ul of caffeine and theophylline riboswitches to separate tubes. I also did a control with just plasmid. I added 1ul of water in place of the riboswitch for the control.
		+	I put the tubes into the PCR machine.  I program it for the following cylces:
		+	▪ 20 cycles of 37C for 1 minute/16C for 1 minute
		+	▪ 1 cycle of 37C for 15 minutes
		+	▪ 22C holding temperature
		+	After the cycles are finished I will go straight to transformation.
		+
		+	Transformation of Riboswitches:
		+	1) I thawed the zippy competent cells on ice for 5 minutes. Each tube contains 50 µL of cells.
		+	2) I aliquot 50ul of cells into a cold test tube
		+	3) I added 10µL of GGA ligation mixture
		+	4) I incubated the ligation and cell mixture on ice for 5 minutes.
		+	5) I Add 40ul of SOC media to a final volume of 100 µL.
		+	6) I spread the cells onto LB Amp plates
		+	7) I placed the plates in a 37C incubator overnight
		+
		+	Tomorrow I will perform a colony PCR to verify if the ligation and transformation were successful.
		+
		+	1-23-14
		+
		+	I check the transformations. The control had three small colonies. Caffeine and theophylline riboswitch both had one very small colony. Either it needs more time to grow or the plates are bad. I will let them grow a little longer. The plates were made in September and the antibiotic resistance might be gone. I can check the plates by just plating some cells on them and see if they grow today. I might need to make more plates.
		+
		+	I talked to Dr. Campbell and I might have had to much insert for the ratio to plasmid. I had about 3:1 ration of insert to plasmid. GGA was originally designed for 1:1. The insert is about 200bp and the plasmid is about 3000bp. 3000/200 is 16%. My plasmid is 50ng/ul so if divide 16/5 I get 3.2. I want a 1/1 so if I divide 10ng/ul be 3.2 I get that I need 3.2ng of insert to 50ng of plasmid to have a 1/1 ratio by weight.
		+
		+	It is also possible that the TE could be causing problem, since it contains EDTA.
		+
		+	Dilution of riboswitches to 3.2ng of insert:
		+	I took 1.6ul of both the caffeine and theophylline riboswitch and mixed with 3.4ul of water.
		+
		+	GGA of Caffeine and Theophylline riboswitch with J100079 Plasmid
		+
		+	1 µL (50 ng) plasmid containing J100079
		+	5µL dH2O
		+	1 µL 10X Promega Ligase Buffer
		+	1 µL 500 mM KOAc
		+	0.5 µL Bsa I high fidelity (HF) restriction enzyme
		+	0.5 µL T4 DNA Ligase
		+	9 µL final volume
		+
		+	I added 1 ul of 3.2ng/ul of caffeine and theophylline riboswitches to separate tubes. I also did a control with just plasmid. I added 1ul of water in place of the riboswitch for the control.
		+	I put the tubes into the PCR machine.  I program it for the following cylces:
		+	▪ 20 cycles of 37C for 1 minute/16C for 1 minute
		+	▪ 1 cycle of 37C for 15 minutes
		+	▪ 22C holding temperature
		+	After the cycles are finished I will go straight to transformation.
		+
		+	Transformation of Riboswitches:
		+	1) I thawed the zippy competent cells on ice for 5 minutes. Each tube contains 50 µL of cells.
		+	2) I aliquot 50ul of cells into a cold test tube
		+	3) I added 10µL of GGA ligation mixture
		+	4) I incubated the ligation and cell mixture on ice for 5 minutes.
		+	5) I Add 40ul of SOC media to a final volume of 100 µL.
		+	6) I spread the cells onto LB Amp plates
		+	7) I placed the plates in a 37C incubator overnight
		+
		+	Tomorrow I will perform a colony PCR to verify if the ligation and transformation were successful.

@@ Line 2,108: / Line 2,108: @@
 Phylogenetic Tree:
-[[File: phylogenetictreetheophylline.png]]
+[[File:phylogenetictreetheophylline.png]]
 There are four groups 1) 1, 2 2) 8,3 3) 6,7 4), 4,5. 3 and 8 are outliers.
@@ Line 2,134: / Line 2,134: @@
 Phylogenetic Tree:
-[[File: phylogenetictreecaffeine.png]]
+[[File:phylogenetictreecaffeine.png]]

@@ Line 2,105: / Line 2,105: @@
 .	ggugauaccagcaucgucuugaugcccuuggcagcacccugcuaagguAACGUGAAGaug
-[[File:guidetreetheophylline.jpg]]
+[[File:guidetreetheophylline.png]]
 Phylogenetic Tree:
-[[File: phylogenetictreetheophylline.jpg]]
+[[File: phylogenetictreetheophylline.png]]
 There are four groups 1) 1, 2 2) 8,3 3) 6,7 4), 4,5. 3 and 8 are outliers.
@@ Line 2,131: / Line 2,131: @@
 I put the 17 sequences of caffeine into clustalW and the guide and phylogenetic Tree:
 Guide Tree:
-[[File:guidetreecaffeine.jpg]]
+[[File:guidetreecaffeine.png]]
 Phylogenetic Tree:
-[[File: phylogenetictreecaffeine.jpg]]
+[[File: phylogenetictreecaffeine.png]]

@@ Line 2,221: / Line 2,221: @@
 Below is the design for cloning the synthesized riboswitch for theophylline into the reporter construct.
-[[File:riboswitchdesign.jpg]]
+[[File:riboswitchdesign.png]]
@@ Line 2,281: / Line 2,281: @@
 >>>Self dimer ΔG: -6.5 kcal.mole-1
 >>>Hairpin ΔG: -1.6 kcal.mole-1
 ==[[User:Catherine|cadoyle]], 14 January 2014==

@@ Line 2,286: / Line 2,286: @@
 My objective today is to design a way to clone the caffeine riboswitches. I was thinking that similar to how I planned on cloning the theophylline riboswitch into the reporter construct I could design the riboswitch to synthesized. Then I would design unique primers to change the individual bases that differ between the putative riboswitches. Then you could do one iPCR reaction for each riboswitch and preform GGA. See Design Below:
-[[File:primerdesign.jpg]]
+[[File:primerdesign.png]]
 I talked to Dr. Campbell and my design will work expect a faster way to screen would be to add a PST site so if GGA worked than the riboswitch will not have a PST site and we do not need to send for sequencing every time. I am going to design both the caffeine and theophylline riboswitch to be synthesized and then start working on primers for 17 caffeine switches. I fixed the theophylline primers see above (1-13-14) and riboswitch to be sequenced.
 ==[[User:Catherine|cadoyle]], 15-16 January 2014==

← Older revision		Revision as of 19:07, 6 December 2013
Line 2,034:		Line 2,034:
	==[[User:Catherine\|cadoyle]], 6 December 2013==		==[[User:Catherine\|cadoyle]], 6 December 2013==
	For some reason running just Riboswitch D did not work. When I added one before it worked, but the first switch in the file is not being read. I know that it writes it to a file but it does not open it dg file. I am not sure or how to fix it. I tired placing multiple riboswitches in a file but only one was read. Maybe it is not reading on all the switches. I am not sure. I printed each line in the program but could not figure out what was going on.		For some reason running just Riboswitch D did not work. When I added one before it worked, but the first switch in the file is not being read. I know that it writes it to a file but it does not open it dg file. I am not sure or how to fix it. I tired placing multiple riboswitches in a file but only one was read. Maybe it is not reading on all the switches. I am not sure. I printed each line in the program but could not figure out what was going on.
		+
		+
		+	[[File: labpresentation12-6-13.pptx]]

← Older revision		Revision as of 13:46, 6 December 2013
Line 2,031:		Line 2,031:

	I read online that if you write wb when you create a file open("filename", wb) instead of w that extra characters are removed. I did this and it worked the carriage return was not present. I ran the script but still got the wrong delta G. I noticed there was a weird space in the output. I printed line by line and found that when I split the lines the was a blank line. I removed the line hoping that might fix the problem but it does not work. I still get the wrong answers. I started playing with the number of switches in a file and the delta G of riboswtich D changed depending on how many switches. I didn't have time to finish but will pick up here tomorrow.		I read online that if you write wb when you create a file open("filename", wb) instead of w that extra characters are removed. I did this and it worked the carriage return was not present. I ran the script but still got the wrong delta G. I noticed there was a weird space in the output. I printed line by line and found that when I split the lines the was a blank line. I removed the line hoping that might fix the problem but it does not work. I still get the wrong answers. I started playing with the number of switches in a file and the delta G of riboswtich D changed depending on how many switches. I didn't have time to finish but will pick up here tomorrow.
		+
		+	==[[User:Catherine\|cadoyle]], 6 December 2013==
		+	For some reason running just Riboswitch D did not work. When I added one before it worked, but the first switch in the file is not being read. I know that it writes it to a file but it does not open it dg file. I am not sure or how to fix it. I tired placing multiple riboswitches in a file but only one was read. Maybe it is not reading on all the switches. I am not sure. I printed each line in the program but could not figure out what was going on.