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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).
		+
		+
		+	*[[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.

---

Revision as of 13:40, 8 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).

• 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

13 Aug 2007

14 Aug 2007

15 Aug 2007

16 Aug 2007

17 Aug 2007

18 Aug 2007