"

From 2012.igem.org

Revision as of 23:55, 3 October 2012 (view source) Ajl58 (Talk | contribs) (Created page with "====I. Finished concentration and dilution calculations==== ====II. Made all diluted (25 mM) primer solutions==== ====III. Ran a 7-8 mutation PCR, but forgot to set melting tempe...") ← Older edit		Latest revision as of 23:56, 3 October 2012 (view source) Ajl58 (Talk | contribs)
Line 1:		Line 1:
-	====I. Finished concentration and dilution calculations====	+	====I. 15-16, 29-30 mutations at 61C melting temperature====
-	====II. Made all diluted (25 mM) primer solutions====	+	====II. 9-10 and retried 7-8 at 55C melting temperature====
-	====III. Ran a 7-8 mutation PCR, but forgot to set melting temperature as 5-10 degrees lower than TM====	+	====III. 21-22 at 67C melting temperature====
-	====IV. Tried to figure out how to make protein gels====	+	====IV. Meeting with Prof. Isaacs====
		+	====V. Observed and recorded results of the purple experiment:====
		+
		+	[[File:Andriana_Notebook_9.jpg|center|thumb|700px]]
		+
		+	{| class="wikitable"
		+	|-
		+	! Plate !! Properties
		+	|-
		+	| 2 A || 150 colonies
		+	|-
		+	| 2 B || 400 colonies
		+	|-
		+	| 2 C || 430 colonies
		+	|-
		+	| 2 D || 900 colonies
		+	|-
		+	| 4 A || 18 colonies
		+	|-
		+	| 4 B || 30 colonies
		+	|-
		+	| 4 C || 300 colonies
		+	|-
		+	| 4 D || 800 colonies
		+	|-
		+	|}
		+
		+	*Analysis
		+
		+	:*Freezing at -80C does increase E. coli death rates
		+	:*Greatest E. coli death was observed at .1M NaCl, so optimal concentration for cell death is less than 1M but more than .01M NaCl.
		+	:*All things said, all data points to the fact that freezing in salt solutions does not provide an effective selection pressure for finding mutants with the most effective AFP or that produce large amounts of AFP because the normal, untransformed control cells exhibited greater survival rates than their transformed counterparts
		+	:*The purple experiment showed that antibiotic plates alone did not cause the reduced colony numbers in the red experiment because the trend of lesser survival of cells 3 and 4 also persisted in the purple experiment
		+	====VI. Digested PCR reactions with DPN1:====
		+	#Add 1 microliter of DPN1 for 50microliters of reaction
		+	#Incubate solution at 37C for an hour
		+	#Heat kill DPN1 enzymes at 80C for 20 minutes

---

Latest revision as of 23:56, 3 October 2012

Contents 1 I. 15-16, 29-30 mutations at 61C melting temperature 2 II. 9-10 and retried 7-8 at 55C melting temperature 3 III. 21-22 at 67C melting temperature 4 IV. Meeting with Prof. Isaacs 5 V. Observed and recorded results of the purple experiment: 6 VI. Digested PCR reactions with DPN1:

I. 15-16, 29-30 mutations at 61C melting temperature

II. 9-10 and retried 7-8 at 55C melting temperature

III. 21-22 at 67C melting temperature

IV. Meeting with Prof. Isaacs

V. Observed and recorded results of the purple experiment:

Plate	Properties
2 A	150 colonies
2 B	400 colonies
2 C	430 colonies
2 D	900 colonies
4 A	18 colonies
4 B	30 colonies
4 C	300 colonies
4 D	800 colonies

• Analysis

• Freezing at -80C does increase E. coli death rates
• Greatest E. coli death was observed at .1M NaCl, so optimal concentration for cell death is less than 1M but more than .01M NaCl.
• All things said, all data points to the fact that freezing in salt solutions does not provide an effective selection pressure for finding mutants with the most effective AFP or that produce large amounts of AFP because the normal, untransformed control cells exhibited greater survival rates than their transformed counterparts
• The purple experiment showed that antibiotic plates alone did not cause the reduced colony numbers in the red experiment because the trend of lesser survival of cells 3 and 4 also persisted in the purple experiment

VI. Digested PCR reactions with DPN1:

1. Add 1 microliter of DPN1 for 50microliters of reaction
2. Incubate solution at 37C for an hour
3. Heat kill DPN1 enzymes at 80C for 20 minutes