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		+	*Minipreppred OMP-YFP sent off for sequencing. 10ul of miniprep and 2ul each of [[Melbourne/primary vf2|vf2]] and [[Melbourne/primary vR|vR]] primers (10uM) were sent.
		+	Sequence was the desired [[Melbourne/BBa_R0082|P1 15P]] to [[Melbourne/BBa_E0430|P1 11A]] ligation product (ampR).
		+
		+
		+
		+	<font color=green size=3>
		+	Part created:
		+	[[Melbourne/OMP-EYFP|OMP-EYFP]]</font>
		+
		+
		+
		+	*[[Melbourne/Growing up cells|Liquid cultured]] [[Melbourne/BBa_J61035|P4 8J]] and [[Melbourne/BBa_E0033|P2 9E]] (from 1/8) in Kan.
	<font size=3><b>6 Aug 2007		<font size=3><b>6 Aug 2007
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		+
		+	*[[Melbourne/Miniprep protocol|Miniprepped]] and *[[Melbourne/Making glycerol Stocksl|created Glycerol Stocks]] of cultures from 5/8.
		+	*[[Melbourne/Diagnostic Digest|Digested]]:
		+	*# [[Melbourne/BBa_E0033|P2 9E]] E/P for 1.5h
		+	*# [[Melbourne/BBa_E0033|P2 9E]] E/X for 2.5h
		+	*# [[Melbourne/BBa_J61035|P4 8J]] E/S for 2.5h
		+
		+	*[[Melbourne/Loading a DNA gel|Ran on a Gel]]:
		+	*# [[Melbourne/primary DNA marker|DNA ladder]]
		+	*# [[Melbourne/BBa_J61035|P4 8J]] E/S
		+	*# [[Melbourne/BBa_J61035|P4 8J]] E/S
		+	*# [[Melbourne/BBa_E0033|P2 9E]] E/P
		+	*# [[Melbourne/BBa_E0033|P2 9E]] E/P
		+	*# [[Melbourne/BBa_E0033|P2 9E]] E/X
		+	*# [[Melbourne/BBa_E0033|P2 9E]] E/X
		+	*# [[Melbourne/primary DNA marker|DNA ladder]]
		+
		+	-All above samples seem to be good.
		+	*Heat inactivated [[Melbourne/BBa_E0033|P2 9E]] E/X and [[Melbourne/BBa_J61035|P4 8J]] E/S at 65degreesC for 10min.
		+
		+	*[[Melbourne/Ligation Protocol|Ligated]] above DNA fragments together (5ul each) at 4degreesC overnight.
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		+
		+	*[[Melbourne/Transformation Protocol|Transformed]] [[Melbourne/BBa_Q04510|P2 13K]] from iGEM plate =CI. Also transformed ligations from 6/8 (GenR-LacZa) = L.
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		+	*[[Melbourne/Growing up cells|Cultured]] transformants CI 1-4 and L1-3 from above transformations.
	<font size=3><b>14 Aug 2007		<font size=3><b>14 Aug 2007
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		+	*[[Melbourne/Miniprep protocol|Miniprepped]] CI-2,3,4. CI-1 and L1-3 showed no growth.
		+	*[[Melbourne/Diagnostic Digest|Digested]] miniprepped DNA for 1.5h:
		+	*# [[Melbourne/primary DNA marker|DNA ladder]]
		+	*# CI-2 E/S
		+	*# CI-2 X/S
		+	*# CI-3 E/S
		+	*# CI-3 X/S
		+	*# CI-4 E/S
		+	*# CI-4 X/S
		+	*# [[Melbourne/primary DNA marker|DNA ladder]]
		+
		+	-CI-2 seems to have the correct DNA.
		+	*[[Melbourne/Making glycerol Stocksl|Glycerol Stock]] of CI-2 created.
	<font size=3><b>15 Aug 2007		<font size=3><b>15 Aug 2007
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		+	*[[Melbourne/Diagnostic Digest|Digests]] of CI-2 and a double terminator ([[Melbourne/BBa_B0014|P1 1G]]) were set up.
		+	*# CI-2 E/S
		+	*# [[Melbourne/BBa_B0014|P1 1G]] E/X
		+
		+	and incubated for 2h at 37degreesC.
		+
		+	*Gel purified 1.8kb band from CI-2 digest and 3kb band from [[Melbourne/BBa_B0014|P1 1G]] digest. These were eluted in 45ul TE buffer.
		+	*These fragments were then [[Melbourne/Ligation Protocol|Ligated]] together using 5ul of [[Melbourne/BBa_B0014|P1 1G]] vector and 7ul of CI-2 insert, overnight at 4degreesC.
	<font size=3><b>18 Aug 2007		<font size=3><b>18 Aug 2007
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		+
		+	*[[Melbourne/Transformation Protocol|Transformation]] of above ligation (17/8) onto a Amp + Kan plate. Plate labeled "ligation."

---

Latest revision as of 07:56, 9 October 2007

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Contents 1 Week 5 2 Week 6 3 Week 7 4 Week 8

Week 5

23 July 2007

• Colony PCRed using vf2 and vR primers the transformations from 20/7. 3 colonies from ligation plate and one from plasmid control plate.

24 July 2007

• Ran Gel from PCRs (23/7) at 100V.

-results: colony 3 didn't give product.

• Streaked out some cells from index plate (No.3).

25 July 2007

26 July 2007

27 July 2007

Week 6

30 July 2007

• Digested minipreprs from 9/7:
  1. P1 11A with X/P
  2. P1 15P with S/P

(no BSA or AP used).

• Inactivated enzymes at 80degreesC in a heat block for 10min.
• Ligated the two parts together (5ul each) overnight at 4degreesC.

31 July 2007

• Transformation of ligations from 31/7 into NM522 competent cells using 3ul of DMSO.

• Diagnostic Digest of
  1. P2 21B
  2. P2 23N
  3. P3 20I

using miniprep from 13/7. All digests were E/P.

Ran a Gel:

• 1. DNA ladder
  2. P2 21B
  3. P2 21B
  4. P2 23N
  5. P2 23N
  6. P3 20I
  7. P3 20I

-Results:

• 2. Low digestion efficiency
  3. Low digestion efficiency
  4. No insert seen but DNA present
  5. No insert seen but DNA present
  6. Ok digestion (not very good)
  7. Ok digestion (not very good)

--Digestion imcomplete??

1 Aug 2007

• Cultured 6 colonies picked from 1/8 transformations. A total of about 500 colonies grew with none on control (competent cells only plate).

• Transformation of P2 9E (LacZa with KanR) from iGEM plate.

2 Aug 2007

• Alkaline Lysis Miniprepped colonies from 1/8. (P1 11A + P1 15P ligation. Named OMP-YFP)

-results: colony 6 had two bands of sizes approximately 1000 and 1100bp.

• Streaked out colony 6 liquid culture on LBA plate and grew overnight.

3 Aug 2007

• Picked 5 colonies from streaked out plate from 2/8 and Cultured overnight.

4 Aug 2007

• Alkaline Lysis Miniprepped colonies from 3/8.

-All lanes had a single band at 1000-1100bp. Two had a 1000bp band and three a 1100bp band.

• Colony 3 of above (1100bp band) was Miniprepped using the Wizerd kit.
• OD of miniprep measured using a spectrophotometer to be 20ng/ul.

Week 7

5 Aug 2007

• Minipreppred OMP-YFP sent off for sequencing. 10ul of miniprep and 2ul each of vf2 and vR primers (10uM) were sent.

Sequence was the desired P1 15P to P1 11A ligation product (ampR).

Part created: OMP-EYFP

• Liquid cultured P4 8J and P2 9E (from 1/8) in Kan.

6 Aug 2007

• Miniprepped and *created Glycerol Stocks of cultures from 5/8.
• Digested:
  1. P2 9E E/P for 1.5h
  2. P2 9E E/X for 2.5h
  3. P4 8J E/S for 2.5h

• Ran on a Gel:
  1. DNA ladder
  2. P4 8J E/S
  3. P4 8J E/S
  4. P2 9E E/P
  5. P2 9E E/P
  6. P2 9E E/X
  7. P2 9E E/X
  8. DNA ladder

-All above samples seem to be good.

• Heat inactivated P2 9E E/X and P4 8J E/S at 65degreesC for 10min.

• Ligated above DNA fragments together (5ul each) at 4degreesC overnight.

7 Aug 2007

8 Aug 2007

9 Aug 2007

10 Aug 2007

11 Aug 2007

Week 8

12 Aug 2007

• Transformed P2 13K from iGEM plate =CI. Also transformed ligations from 6/8 (GenR-LacZa) = L.

13 Aug 2007

• Cultured transformants CI 1-4 and L1-3 from above transformations.

14 Aug 2007

• Miniprepped CI-2,3,4. CI-1 and L1-3 showed no growth.
• Digested miniprepped DNA for 1.5h:
  1. DNA ladder
  2. CI-2 E/S
  3. CI-2 X/S
  4. CI-3 E/S
  5. CI-3 X/S
  6. CI-4 E/S
  7. CI-4 X/S
  8. DNA ladder

-CI-2 seems to have the correct DNA.

• Glycerol Stock of CI-2 created.

15 Aug 2007

16 Aug 2007

17 Aug 2007

• Digests of CI-2 and a double terminator (P1 1G) were set up.
  1. CI-2 E/S
  2. P1 1G E/X

and incubated for 2h at 37degreesC.

• Gel purified 1.8kb band from CI-2 digest and 3kb band from P1 1G digest. These were eluted in 45ul TE buffer.
• These fragments were then Ligated together using 5ul of P1 1G vector and 7ul of CI-2 insert, overnight at 4degreesC.

18 Aug 2007

• Transformation of above ligation (17/8) onto a Amp + Kan plate. Plate labeled "ligation."