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	*[[Melbourne/BBa_J61035|P4 8J]] and [[Melbourne/BBa_C0051|P1 5G]] were overgrown so were streaked onto new plates.		*[[Melbourne/BBa_J61035|P4 8J]] and [[Melbourne/BBa_C0051|P1 5G]] were overgrown so were streaked onto new plates.
-	*[[Melbourne/Colony PCRl|Colony PCR]] using [http://openwetware.org/wiki/Endy:Colony_PCR]	+	*[[Melbourne/Colony PCRl|Colony PCR]] using The Endy Procol [http://openwetware.org/wiki/Endy:Colony_PCR] of 2 colonies from ComA-cell death (12/9) and OmpR-L3 (27/8),
		+
		+	*# 5ul 10 X PCR buffer (green)
		+	*# 5ul MgCl2 25mM
		+	*# 0.5ul [[Melbourne/primary vf2|vf2]]
		+	*# 0.5ul [[Melbourne/primary vR|vR]]
		+	*# 1.0ul dNTPs 25mM
		+	*# 0.5ul GoTaq
		+	*# 37.5ul [[Melbourne/primary milliq|milliQ water]]
		+
		+	on a cycle of:
		+	*# 94C for 5min
		+	*# 94C for 30 seconds
		+	*# 55C for 30 seconds
		+	*# 72C for 1:15 minutes
		+	*# repeat 2-4 for 30 times
		+	*# 72C for 7min.
	<font size=3><b>14 Sept 2007		<font size=3><b>14 Sept 2007

---

Revision as of 12:12, 9 October 2007

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Contents 1 Week 9 2 Week 10 3 Week 11 4 Week 12

Week 9

19 Aug 2007

• Picked 3-4 transformants from "ligation" plates (17/8) and Grew up liquid cultures.
• Transformed the two ligation reactions in the pink rack in the cold room and plated on Gen + Amp plate.

20 Aug 2007

• Miniprepped liquid cultures from 19/8 labeled LA, LB, LC and P2 13K A, P2 13K B.
• Concentration of DNA was measured to be:
  A- 221ng/ul
  B-224ng/ul.
  

• Digested:
  1. LA X/S
  2. LB X/S
  3. LC X/S
  4. P2 13K A X/S
  5. P2 13K B X/S

Desired products:

• 1. 450bp (95bp different from CI-2 used as control)
  2. 450bp
  3. 450bp
  4. 1kb
  5. 1kb

• These were run on a gel.

• Digestions were set up for ligations.

OmpR-inverter
-Vector: OmpR (AmpR) miniprepped P1 15P from 10/7 digested with S/P.
-Insert: c1 inverter (1kb fragment) P2 13K A with X/P.

RBS-LacZ
-Vector: LacZ (KanR) P2 9E from 6/8 with E/X.
-Insert: RBS (GenR) P4 8J from 6/7 with E/S.

Digested for 2h and heat inactivated for 10min at 80degC.

• Made Glycerol Stocks of P2 13K using dry ice method.

21 Aug 2007

• Picked 4 colonies from fresh ligation/transformation to Growing up.

- These transformations were made as follows:

• • Ligated
    1. 5ul of Purified insert from 17/8 (CI-2 E/S)
    2. 5ul of Redigested P1 5G with E/X and heat inactivated at 60degC for 10min.

• Recieved M30109 and CP919.

22 Aug 2007

• Miniprepped L1-L4 (liquid cultures from 21/8).
• Digested these for ligations.
  1. L1 X/P
  2. L2 X/P
  3. L3 X/P
  4. L4 X/P
  5. CI-2 X/P (control for 350bp)
  6. P1 15P S/P

• The above were Ran on a Gel together with P2 13K X/P digest from 21/8.

All L1-L4 had the 450bp desired band.

• Gel purified 450bp band from L3 and a 950bp band from the P2 13K digest.

• Ligated the following:

OmpR-L3 (RBS-LacZa-double term)
-Vector= P1 15P S/P (above)
-Insert= L3 X/P (above)

OmpR-c1 inverter (P2 13K)
-Vector= P1 15P S/P (above)
-Insert= P1 15P S/P (above)

Ligations were kept overnight at 4degC.

23 Aug 2007

• Transformed ligations from 22/8 into competent NM522 cells.

24 Aug 2007

• Liquid cultures were made of transformations from 23/8. 6 colonies were picked from each ligation.

25 Aug 2007

• Miniprepped cultures from 23/8 and M30109 cells.
• Digested:
  • 12 minipreps from ligations X/P
  • 2 minipreps from M30109 cells S/P
  • 2 minipreps from M30109 cells X/P

• Ran on a Gel 16 samples that were miniprepped.
• Gel purified 1000bp fragment from ompR-c1 inverter ligation digest and a 600bp fragment from ompR-L3 ligation digest.

Week 10

26 Aug 2007

• Ligated
  1. M30109 S/P to ompR-L3 X/P
  2. M30109 S/P to ompR-c1 inv X/P

(all from 25/8) Left overnight at 4degC.

27 Aug 2007

• Transformed
  1. M30109 S/P to ompR-L3 X/P (26/8)
  2. M30109 S/P to ompR-c1 inv X/P (26/8)
  3. P1 15P-L3 (from 22/8)
  4. P1 15P-c1 inverter (from 22/8)

Plated on Amp plates.

28 Aug 2007

• Picked six colonies from each plate (27/8) and cultured at 37degC for 13 hours.

29 Aug 2007

• Miniprepped 24 cultures from 29/8.
• Diagnosted all with X/S.
• Ran on a Gel

-The M30109 ligations yielded random fragments: something wrong with the part? - P1 15P-L3 and P1 15P-c1 inverter ligations had bands of the desired size.

30 Aug 2007

• Set up minipreps from 29/8 for sequencing.

OmpR-L3 and OmpR-c1 inverter were the desired sequences.

Parts created:

OmpR-RBS-LacZa-double terminator

OmpR-c1 inverter

31 Aug 2007

1 Sept 2007

Week 11

2 Sept 2007

• Digested and Ran on a Gel M30109 minipreps from 25/8 using a number of restriction enzymes to try to find out what it is:

• 1. EcoRV
  2. Xho1
  3. EcoRV/Xho1
  4. E/P
  5. E/S
  6. E/X
  7. X/P
  8. X/S
  9. S/P
  10. E
  11. P
  12. S
  13. EcoR1/EcoRV/Xho1
  14. Apa1
  15. BamH1
  16. EcoR1/BamH1

The results showed that although a fragment of cph8 may be present in the part (1200bp fragment present only when double digested by EcoRV/Xho1), it may not be in the form as designated in the iGEM wiki.

The original miniprep was sent off for sequencing using vf2. The results showed that the part is a form of RBS-cph8, although the sequencing got cut off in the middle of the cp8 sequence. Therefore, the entire part designated is not present. We consequently decided to create the M30109 part ourselves.

3 Sept 2007

4 Sept 2007

• Made LB (600ml) + LB-agarose (400ml)
• Created Agar Plates with Kan.

5 Sept 2007

• Recieved ComP and ComA sequences from GeneArt.

6 Sept 2007

• Transformed the following on Amp plates except where labeled
  1. ComA
  2. ComP (Kan plate)
  3. 6kb band from undigested M30109
  4. 10kb band from undigested M30109
  5. RBS-LacZa-double terminator from 22/8
  6. OmpR-RBS-LacZa-double terminator from 29/8
  7. OmpR-RBS-LacZa-double terminator from 29/8 into CP919 EnvZ- strains.

7 Sept 2007

• Liquid cultured a colony from each of the 6/9 transformations. No growth was observed for the M30109 undigested plates and were discarded.

8 Sept 2007

• Miniprepped cultures from 7/9.
• Digested these with X/P
• Ran these on a Gel.

-All had desired sizes.

• Glycerol Stocks of all four of these were created.

Week 12

9 Sept 2007

• Ligated the following:

ComP-backbone vector
-Vector: P3 20I Death Plasmid AmpR X/P fragment from 8/9.
-Insert: ComP X/P fragment from 8/9.

ComA-backbone vector
-Vector: P3 20I Death Plasmid AmpR X/P fragment from 8/9.
-Insert: ComA X/P fragment from 8/9.

10 Sept 2007

11 Sept 2007

12 Sept 2007

• Transformed ligations from 9/9.
• Also Transformed resuspended DNA for the following:
  1. P2 21A
  2. P2 21C
  3. P48F
  4. P17O
  5. P11I

and minipreprs from previous dates:

• 1. P4 8J from 5/7
  2. P1 5G

• Efficiency of fast ligations were simultaneously tested for. Using 2X ligase buffer, 2hour and 5min ligations were tested using ComA parts from 8/9. These did not have ligation efficiencies as high as the overnight ligations but were sufficient. The 5 min ligation seemed to have more colonies than the 2 hour ligation.

13 Sept 2007

• P4 8J and P1 5G were overgrown so were streaked onto new plates.
• Colony PCR using The Endy Procol [http://openwetware.org/wiki/Endy:Colony_PCR] of 2 colonies from ComA-cell death (12/9) and OmpR-L3 (27/8),

• 1. 5ul 10 X PCR buffer (green)
  2. 5ul MgCl2 25mM
  3. 0.5ul vf2
  4. 0.5ul vR
  5. 1.0ul dNTPs 25mM
  6. 0.5ul GoTaq
  7. 37.5ul milliQ water

on a cycle of:

• 1. 94C for 5min
  2. 94C for 30 seconds
  3. 55C for 30 seconds
  4. 72C for 1:15 minutes
  5. repeat 2-4 for 30 times
  6. 72C for 7min.

14 Sept 2007

15 Sept 2007