IGEM Notebook - Revision history

Olhoshing: /* Thursday, June 4, 2009 */

2009-08-04T05:00:04Z

‎Thursday, June 4, 2009

ShPunjabi: /* Tuesday, June 9, 2009 */

2009-06-18T19:49:50Z

‎Tuesday, June 9, 2009

ShPunjabi: /* Thursday, June 11, 2009 */

2009-06-15T21:54:19Z

‎Thursday, June 11, 2009

ShPunjabi: /* Friday, June 12, 2009 */

2009-06-15T21:52:05Z

‎Friday, June 12, 2009

ShPunjabi: /* Thursday, June 11, 2009 */

2009-06-15T13:09:25Z

‎Thursday, June 11, 2009

ShPunjabi: /* Sunday, June 14, 2009 */

2009-06-15T04:05:12Z

‎Sunday, June 14, 2009

ShPunjabi: /* Sunday, June 14, 2009 */

2009-06-15T04:00:28Z

‎Sunday, June 14, 2009

ShPunjabi: /* Sunday, June 14, 2009 */

2009-06-15T02:57:54Z

‎Sunday, June 14, 2009

ShPunjabi: /* Sunday, June 14, 2009 */

2009-06-15T02:46:35Z

‎Sunday, June 14, 2009

ShPunjabi: /* Sunday, June 14, 2009 */

2009-06-15T02:16:22Z

‎Sunday, June 14, 2009

@@ Line 26: / Line 26: @@
 ==Thursday, June 4, 2009==
-This morning, I began the day by learning how to use the PEARL prgram. Olivia Ho-Shing sat with me and walked me through the program script. And then, Leland Taylor and Shashank Suresh had a problem they wanted us to solve. Leland explained that we need to make sure there is a stop codon in the gene if we fail to suppress the 5mer. The stop codon needs to be before we find another 5mer. They also wanted us to run through both the RFP and Tet Resistance genes to make sure that they did not contain any of the anticodon sequences from our 5 frameshift suppressor tRNAs so as to throw the translation out of frame. They further wanted us to confirm that if we do have successful suppression, there are no stop codons in the middle of the RFP and Tet Resistance genes. PEARL did not find any matches for the suppressor tRNAs within genes. We found 3 matches for stop codons UAA. Two of these occured at the end of the Tet and one at the end of RFP, where they should normally be to end translation into a protein.
+This morning, I began the day by learning how to use the PERL prgram. Olivia Ho-Shing sat with me and walked me through the program script. And then, Leland Taylor and Shashank Suresh had a problem they wanted us to solve. Leland explained that we need to make sure there is a stop codon in the gene if we fail to suppress the 5mer. The stop codon needs to be before we find another 5mer. They also wanted us to run through both the RFP and Tet Resistance genes to make sure that they did not contain any of the anticodon sequences from our 5 frameshift suppressor tRNAs so as to throw the translation out of frame. They further wanted us to confirm that if we do have successful suppression, there are no stop codons in the middle of the RFP and Tet Resistance genes. PERL did not find any matches for the suppressor tRNAs within genes. We found 3 matches for stop codons UAA. Two of these occured at the end of the Tet and one at the end of RFP, where they should normally be to end translation into a protein.
 Following this, Olivia Ho-Shing and I tried to determine how we can add BioBrick ends to our single stranded DNA sequences that code for the suppressor tRNAs. Our ultimate goal is to find complements for these single strands so they can be put into plasmids. We found the standard prefixes and suffixes for sequences that do not contain ATG (becuase this sequence is a functional RNA and will not be translated) and we placed these before and after the altered 92bp sequence that Dr. Anderson provided us with. The prefixes and suffixes are necessary because they are the extra nucleotides that will be mimicking a "restriction enzyme cutting site". The single strand appeared as below after the Bio Brick prefixes and suffixes were added:

@@ Line 230: / Line 230: @@
 The following shows which parts we entered in which gels:
-[[Image: screen-capture-2.png]]
+[[Image: Gel chart June_9.png]]
 We unloaded the 2.5% agarose first and after observing the bands under a UV light, put the gel into the BioRad machine to observe the bands. The image below is of a Molecular Weight Marker that shows the number of bp in control fragments of DNA according to the distance they travel down the gel.

@@ Line 374: / Line 374: @@
 [[Image: Pcrtubes.png]]
-I had
+I had CCAUC as 5mer.
 We followed the temperature cycles so that PCR could occur. The annealing temperature needed to be 5C less than the smallest melting temperature of the primers. Since the reverse primer of RFP had a melting temp of 66C, we set the temperature of the 3rd cycle on 61C.

← Older revision		Revision as of 21:52, 15 June 2009
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−	The other half of the group (which included me) prepared the agarose gels so that we could run gels for the digested reporters and the PCR inserts. We calculated that the digested reporters needed an agarose of 4% and the PCR reporters needed an agarose of 1.45%. We allowed this gel to form for about 45 minutes to an hour. We prepared both the gels and allowed them to run through lunch.	+	The other half of the group (which included me) prepared the agarose gels so that we could run gels for the digested reporters and the PCR inserts. We calculated that the digested reporters needed an agarose of 0.4% and the PCR reporters needed an agarose of 1.45%. We allowed this gel to form for about 45 minutes to an hour. We prepared both the gels and allowed them to run through lunch.
	After lunch, we took pictures of our gels and noticed that the third lane in the PCR gel (third lane from the top in the bottom photo) did not develop a band. The lane was supposed to contain the CUAGU insert for Tetracycline Resistance.		After lunch, we took pictures of our gels and noticed that the third lane in the PCR gel (third lane from the top in the bottom photo) did not develop a band. The lane was supposed to contain the CUAGU insert for Tetracycline Resistance.

@@ Line 373: / Line 373: @@
 [[Image: Pcrtubes.png]]
 We followed the temperature cycles so that PCR could occur. The annealing temperature needed to be 5C less than the smallest melting temperature of the primers. Since the reverse primer of RFP had a melting temp of 66C, we set the temperature of the 3rd cycle on 61C.
@@ Line 396: / Line 398: @@
 . Verify our tRNA inserts and eventually transform these into plasmids.
 . Check our agar plates to see colonies that have replicated our pLac+RBS, pBAD+RBS, and pBAD genes.
 ==Friday, June 12, 2009==

← Older revision		Revision as of 04:05, 15 June 2009
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	We also stored liquid cultures of the colonies that greater than a 2:1 variable:negative control ratio just in case we wanted to use them for mini preps in the future. These included #1 (3 samples), #4 (2 samples), #5 (1 sample), and #6 (2 samples).		We also stored liquid cultures of the colonies that greater than a 2:1 variable:negative control ratio just in case we wanted to use them for mini preps in the future. These included #1 (3 samples), #4 (2 samples), #5 (1 sample), and #6 (2 samples).
		+
		+	'''To Do''':
		+	1. Count colonies obtained from second transformation
		+	2. Clean PCRs: Dig, ligate, transform
		+	3. Run gel for 2 Tet PCRs, clean
		+	4. Gel purify receiving vector

← Older revision		Revision as of 04:00, 15 June 2009
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	After spreading the mixture onto the agar plates, we incubated the plates at 10:56 pm.		After spreading the mixture onto the agar plates, we incubated the plates at 10:56 pm.

−	We also stored liquid cultures of the colonies that greater than a 2:1 variable:negative control ratio just in case we wanted to use them for mini preps in the future.	+	We also stored liquid cultures of the colonies that greater than a 2:1 variable:negative control ratio just in case we wanted to use them for mini preps in the future. These included #1 (3 samples), #4 (2 samples), #5 (1 sample), and #6 (2 samples).

← Older revision		Revision as of 02:57, 15 June 2009
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	60uL of LB media		60uL of LB media

		+	After spreading the mixture onto the agar plates, we incubated the plates at 10:56 pm.
		+
	We also stored liquid cultures of the colonies that greater than a 2:1 variable:negative control ratio just in case we wanted to use them for mini preps in the future.		We also stored liquid cultures of the colonies that greater than a 2:1 variable:negative control ratio just in case we wanted to use them for mini preps in the future.

@@ Line 464: / Line 464: @@
 ==Sunday, June 14, 2009==
 We decided to re-ligate and re-transform all of the tRNA inserts because we did not get very good yields of colonies from the transformations.
@@ Line 470: / Line 472: @@
 [[Image: Trnaligation.png]]
 We also stored liquid cultures of the colonies that greater than a 2:1 variable:negative control ratio just in case we wanted to use them for mini preps in the future.