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	====Transformation====		====Transformation====
	#resuspended the following from Registry plates:		#resuspended the following from Registry plates:
-	**BBa_I15008 - ho1 (P2 21A, Kan)	+	##BBa_I15008 - ho1 (P2 21A, Kan)
-	**BBa_I15009 - PcyA (P2 21C, Kan)	+	##BBa_I15009 - PcyA (P2 21C, Kan)
-	**BBa_R0084 - OmpR positive promoter (P1 11H, Amp)	+	##BBa_R0084 - OmpR positive promoter (P1 11H, Amp)
-	##Punctured foil with pipette tip.	+	#Punctured foil with pipette tip.
-	##Resuspended in 15uL ddH2O.	+	#Resuspended in 15uL ddH2O.
-	##Stored in-20 (after taking 1uL for transformation).	+	#Stored in-20 (after taking 1uL for transformation).
	#[[Melb:Transformation Protocol|Transformed]] into competent DH5alpha cells with shorter incubation times as follows:		#[[Melb:Transformation Protocol|Transformed]] into competent DH5alpha cells with shorter incubation times as follows:
-	**30min on ice after DNA addition	+	##30min on ice after DNA addition
-	**10min on ice after heat shock	+	##10min on ice after heat shock
-	**30min at 37degrees with LB	+	##30min at 37degrees with LB
	===26 June 2007===		===26 June 2007===

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Revision as of 02:15, 3 July 2007

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Contents 1 Week 1 1.1 25 June 2007 1.1.1 Transformation 1.2 26 June 2007 1.2.1 Transformation from Monday 1.2.2 Transformation 1.2.3 Streak plates 1.3 27 June 2007 1.3.1 Transformation 1.3.2 Liquid culture 1.4 28 June 2007 1.4.1 Miniprep 1.4.2 Digest 1.4.3 Liquid culture 1.5 29 June 2007 1.5.1 Miniprep 1.6 30 June 2007 2 Week 2 2.1 2 July 2007 3 Now 3.1 3 July 2007 3.2 4 July 2007 3.3 5 July 2007 3.4 6 July 2007 3.5 Week 3 3.5.1 9 July 2007 3.5.2 10 July 2007 3.5.3 11 July 2007 3.5.4 12 July 2007 3.5.5 13 July 2007 3.6 Week 4 3.6.1 16 July 2007 3.6.2 17 July 2007 3.6.3 18 July 2007 3.6.4 19 July 2007 3.6.5 20 July 2007 3.7 Week 5 3.7.1 23 July 2007 3.7.2 24 July 2007 3.7.3 25 July 2007 3.7.4 26 July 2007 3.7.5 27 July 2007 3.8 Week 6 3.8.1 30 July 2007 3.8.2 31 July 2007 3.8.3 1 Aug 2007 3.8.4 2 Aug 2007 3.8.5 3 Aug 2007

Week 1

25 June 2007

Prepared LB agar plates.

Transformation

1. resuspended the following from Registry plates:
   1. BBa_I15008 - ho1 (P2 21A, Kan)
   2. BBa_I15009 - PcyA (P2 21C, Kan)
   3. BBa_R0084 - OmpR positive promoter (P1 11H, Amp)
2. Punctured foil with pipette tip.
3. Resuspended in 15uL ddH2O.
4. Stored in-20 (after taking 1uL for transformation).
5. Transformed into competent DH5alpha cells with shorter incubation times as follows:
   1. 30min on ice after DNA addition
   2. 10min on ice after heat shock
   3. 30min at 37degrees with LB

26 June 2007

Transformation from Monday

• Transformation of BBa_I15008 and BBa_I15009 failed. No colonies on plates
• Small number of colonies on BBa_R0084 plate.
• Placed in cool room

Transformation

1. Resuspended the following parts in 15uL:
   1. BBa_B0034 (RBS, plate 1 3O, Amp)
   2. BBa_C0051 (C1 repressor, plate 1 5G, Amp)
   3. BBa_B0010 (Terminator, plate 2 3P, Amp)

• Think some DNA may have remained in wells

1. Transformed into competent DH5alpha cells from Joe

Streak plates

1. Streaked the following cells:
   1. PJS010 (from solid agar, Amp)
   2. Fusion protein (from glycerol stock, Amp?)
   3. BBa_I15010 (from solid agar, Kan)

27 June 2007

Transformation

1. Repeated transformation of failed parts from Monday:
   1. BBa_I15008 (Kan)
   2. BBa_I15009 (Kan)

• Used resuspended DNA that was stored on Monday

Liquid culture

1. Prepared 30mL of Amp LB (100ug/mL). Prepared 5mL Kan LB (50mg/mL).
2. Aliquoted 5mL Amp LB into 6 50mL falcon tubes
3. To the Amp LB aliquots single transformed colonies from plates of the following were introduced:
   1. PJS010
   2. Fusion
   3. BBa_B0034
   4. BBa_C0051
   5. BBa_B0010
   6. BBa_R0084
4. To the Kan LB a single colony from the transformation plate of BBa_I15010 was introduced.
5. Cells incubated at 37degrees with shaking overnight.

28 June 2007

Miniprep

1. Minipreped the cultures grown overnight after putting aside 1mL(sterile) for glycerol stocks:
   1. PJS010
   2. Fusion
   3. BBa_B0034
   4. BBa_C0051
   5. BBa_B0010
   6. BBa_R0084
   7. BBa_I15010
2. Stored in -20 freezer

Digest

Liquid culture

1. Cultured the following cells from transformed plates:
   1. BBa_R0084
   2. BBa_I15010
   3. BBa_B0034
   4. BBa_C0051
   5. BBa_I15008
   6. BBa_I15009

29 June 2007

Miniprep

1. Minipreped the cultures grown overnight after putting aside 1mL(sterile) for glycerol stocks:
   1. BBa_R0084
   2. BBa_I15010
   3. BBa_B0034
   4. BBa_C0051
   5. BBa_I15008
   6. BBa_I15009

1. Stored in -20 freezer

30 June 2007

Week 2

2 July 2007

Now

3 July 2007

4 July 2007

5 July 2007

6 July 2007

Week 3

9 July 2007

10 July 2007

11 July 2007

12 July 2007

13 July 2007

Week 4

16 July 2007

17 July 2007

18 July 2007

19 July 2007

20 July 2007

Week 5

23 July 2007

24 July 2007

25 July 2007

26 July 2007

27 July 2007

Week 6

30 July 2007

31 July 2007

1 Aug 2007

2 Aug 2007

3 Aug 2007