Revision as of 20:46, 31 May 2010

Building Parts

May
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August
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September
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10-05-2010

PCR a Kanamycin resistance cassette fragment from p1003 with primers containing either the A/B' ends or the B/A' ends.

Recipe:

1uL p1003 (approx. 1ng)

2.5uL prA_p1003+

2.5uL prB'_p1003-

5uL 10X PCR buffer

1uL 10uM dNTPs

2uL 50uM MgCl~2~

0.5uL Taq polymerase

35.5uL MilliQ H~2~O

Program:

11-05-2010

June 3, 2009

Plasmid transformed = pSB1AC3 (TEST)
- Note: team members were of mixed opinion as to whether the transform was likely due to low availability of DNA - DNA was split between a number of transformation reactions for at least 2 different DB3.1 strains. Plates incubated at 30 degrees Celsius overnight, temp. raised to 37 degrees approx. 10:30 a.m.

June 5, 2009

Tet plates made
Recipe for 200 ml (approx. 10 plates):
2.2 g agar in 200 ml fresh LB
Note: do not re-autoclave LB, it will caramelize!

Recipe for 200 ml LB:
a) 1 g yeast extract
b) 2 g peptotryptone
c) 2 g NaCl
d) 200 ml water
Autoclaved at 11:20 a.m., picked up at 1:15 p.m. and cooled in water bath at 37 degrees Celsius
Added 200 microlitres tetracycline stock (from -20 freezer) to final concentration of 20 microgram/ml
Swirl and poured into prepared, labeled plates
- Note: label on plates reads June 3, 2009 but should be June 5, 2009 (8 plates in total - either I overpoured or these plates are a bit larger than I was used to)
Inverted and put in 37 degree incubator to dry

June 8, 2009

Bacteria grown to log phase using sterile inoculating loop to inoculate approx. 200 ml of LB media with bacteria from 2 plates (approx. 3 colonies)
Bacterial liquid culture placed in shaker at 10:51 a.m.

June 9, 2009

Digested miniprepped gel with EcoRI and SpeI
Running gel to see if correct plasmid was purified - should expect to see bands at ~3200 bp
DNA Ladder made - 6 microlitres of stock used

@@ Line 32: / Line 32: @@
 =11-05-2010=
+=June 3, 2009=
+#Plasmid transformed = pSB1AC3 (TEST)
+#*Note: team members were of mixed opinion as to whether the transform was likely due to low availability of DNA - DNA was split between a number of transformation reactions for at least 2 different DB3.1 strains. Plates incubated at 30 degrees Celsius overnight, temp. raised to 37 degrees approx. 10:30 a.m.
+=June 5, 2009=
+#Tet plates made
+#:Recipe for 200 ml (approx. 10 plates):
+#::2.2 g agar in 200 ml fresh LB
+#::Note: do not re-autoclave LB, it will caramelize!
+#:Recipe for 200 ml LB:
+#::a) 1 g yeast extract
+#::b) 2 g peptotryptone
+#::c) 2 g NaCl
+#::d) 200 ml water
+#Autoclaved at 11:20 a.m., picked up at 1:15 p.m. and cooled in water bath at 37 degrees Celsius
+#Added 200 microlitres tetracycline stock (from -20 freezer) to final concentration of 20 microgram/ml
+#Swirl and poured into prepared, labeled plates
+#*Note: label on plates reads June 3, 2009 but should be June 5, 2009 (8 plates in total - either I overpoured or these plates are a bit larger than I was used to)
+#Inverted and put in 37 degree incubator to dry
+=June 8, 2009=
+#Bacteria grown to log phase using sterile inoculating loop to inoculate approx. 200 ml of LB media with bacteria from 2 plates (approx. 3 colonies)
+#Bacterial liquid culture placed in shaker at 10:51 a.m.
+=June 9, 2009=
+#Digested miniprepped gel with EcoRI and SpeI
+#Running gel to see if correct plasmid was purified - should expect to see bands at ~3200 bp
+#DNA Ladder made - 6 microlitres of stock used

Team:Alberta/Building Parts

From 2010.igem.org