From 2007.igem.org

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Contents 1 Week 1 2 Week 2 3 Week 3 4 Week 4

Week 1

• 25 June 2007: Prepared LB agar plates Amp & Kana.
• 25 June 2007: Resuspended the following from Registry plates & Transformed (shorter protocol) into competent DH5alpha cells.
  1. P2 21A - (BBa_I15008, ho1, Kan) -> No colonies on plates
  2. P2 21C - (BBa_I15009, PcyA, Kan) ->No colonies on plates
  3. P1 11H - (BBa_R0084, OmpR positive promoter, Amp)->Small number of colonies.

• 26 June 2007: Resuspended the following from Registry plates & Transformed into Joe's competent DH5alpha cells.
  1. P1 3O (BBa_B0034, RBS, Amp)
  2. P1 5G (BBa_C0051, c1 protein, Amp)
  3. P2 3P (BBa_B0010, Terminator, Amp)

• 26 June 2007: 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: Transformed into Joe's competent DH5alpha cells.
  1. P2 21A (Kan)
  2. P2 21C (Kan)

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:
   • pJS010
   • Fusion
   • P1 3O
   • P1 5G
   • P2 3P
   • P1 11H
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:
   • pJS010
   • Fusion
   • P1 3O
   • P1 5G
   • P2 3P
   • P1 11H
   • I15010
2. Stored in -20 freezer

Liquid culture

1. Cultured the following cells from transformed plates:
   • P1 11H (BBa_R0084)
   • BBa_I15010
   • P1 3O (BBa_B0034)
   • P1 5G (BBa_C0051)
   • P1 21A (BBa_I15008)
   • P1 21C (BBa_I15009)

29 June 2007
Miniprep

1. Minipreped the cultures grown overnight after putting aside 1mL(sterile) for glycerol stocks:
   • P1 11H (BBa_R0084)
   • BBa_I15010
   • P1 3O (BBa_B0034)
   • P1 5G (BBa_C0051)
   • P1 21A (BBa_I15008)
   • P1 21C (BBa_I15009)

1. Stored in -20 freezer

Week 2

• 2 July 2007: Resuspended the following from Registry plates & Transformed into Joe's competent DH5alpha cells.
  1. Q04510
  2. E0241
  3. E0040
  4. B0014
  5. J61035

• 3 July 2007: Re Transformed into Joe's competent DH5alpha cells. Each Plate had additional 40uL of 50mg/ml Ampicillan spread on surface.
  1. P4 8J -> Three colonies -> grew in liquid culture 4 july
  2. P1 5H -> Multiple colonies at edge -> did not grow in overnight liquid culture 4 july 'poor amp spreading'
  3. P2 15L -> Three colonies -> grew in liquid culture 4 july

• 4 July 2007:

Ampicillin Plates

• Made up 25 Ampicillin plates using 400mL LB agar prepared on the 25th of June.
  • Used 100mg/ml Ampicillin stock from Gooley Lab - In bag in left freezer near our lab bench.

Transformation
Transformed the following and grew on new ampicillin plates

• P1 5H
• P4 8J
• P2 15L
  • Also Plated an untransformed control (subjected to transformation protocol in absense of DNA) and a blank plate control (no cells)

Liquid Culture

• Cultured 2 colonies from each of the following transformed plates and labelled as follows
  • P2 3P 1
  • P2 3P 2
  • I15010 1
  • I15010 2
  • pJS010 1
  • pJS010 2
  • Fusion 1
  • Fusion 2
  • P4 8J 1
  • P4 8J 2
  • P2 15L 1
  • P2 15L 2
  • P2 13K 1
  • P2 13K 2
  • P1 1G 1
  • P1 1G 2
  • P1 11H 1
  • P1 11H 2
  • P1 5H 1
  • P1 5H 2
  • suspected contaminating colony from P4 8J
• 4/7/07 Confirmation of Miniprep Plasmids by digestion

5 July 2007

Miniprep

• 1mL culture from the 4th of Julyput aside for glycerol stocks under sterile conditions. Labelled with todays date 5/7
• miniprepped the following overnight cultures set up on the 4th of July. Final elution was performed with TE buffer prepared on 25/06/07 rather than nuclease free water. Labelled with todays date 5/7.
  • P2 3P 1
  • P2 3P 2
  • I15010 1
  • I15010 2
  • pJS010 1
  • pJS010 2
  • Fusion 1
  • Fusion 2
  • P4 8J 1
  • P4 8J 2
  • P2 15L 1
  • P2 15L 2
  • P2 13K 1(eluted in 130uL due to accidental double application of 50uL elution)
  • P2 13K 2
  • P1 1G 1
  • P1 1G 2
• the following liquid cultures were not miniprepped due to failure (no growth)
  • P1 11H 1
  • P1 11H 2
  • P1 5H 1
  • P1 5H 2
  • suspected contaminating colony from P4 8J

Digest
Performed the following digests on DNA from the above miniprep

EcoR1/Pst1 with buffer 3

• I15010 1
• I15010 2
• P4 8J 1
• P4 8J 2
• P2 15L 1
• P2 15L 2
• P2 13K 1
• P2 13K 2

EcoR1/HaeII in buffer 2

• P2 3P 1
• P2 3P 2
• P1 1G 1
• P1 1G 2

XbaI/SpeI in buffer 2

• pJS010 1
• pJS010 2

Incubated for 2hours at 37 degrees, before addition of loading dye and storage at -20

Transformation

• Transformed P1 11H from resuspended DNA.

Liquid Culture

• P1 5H 1
• P1 5H 2
• P4 8J 1
• P4 8J 2
• P2 15L 1
• P2 15L 2

6 July 2007

Digest Gel

• Prepared 20 lane 100mL agarose gel with 0.5xTBE buffer.
• Loaded 20uL of digest samples in the following lane order
  1. 1kb+ ladder
  2. I15010 1
  3. I15010 2
  4. P2 13K 1
  5. P2 13K 2
  6. P2 15L 1
  7. P2 15L 2
  8. P4 8J 1
  9. P4 8J 2
  10. P1 1G 1
  11. P1 1G 2
  12. P2 3P 1
  13. P2 3P 2
  14. pJS010 1
  15. pJS010 2
• Ran for 1.5hours at 95V

Miniprep

• Put aside 1mL of liquid cultures set up on the 5th for glycerol stocks and labelled with todays date 6/7
• Miniprepped the remains of the cultures and labelled with todays date 6/7. Samples were eluted with TE buffer.
• P1 5H 1
• P1 5H 2
• P4 8J 1
• P4 8J 2
• P2 15L 1
• P2 15L 2

Digest

• Digested 5uL of each of the above miniprep DNA with EcoRI and PstI in buffer 3.
• Incubated for 2hours 25min at 37degrees
• Added 5uL 6x loading dye and stored at -20

Glycerol Stocks
The following Glycerol Stocks were made:

• Put aside from cultures 5/7 (labelled with this date)
  1. P2 15L 1
  2. P2 15L 2
  3. P4 8J 1
  4. P4 8J 2
  5. P1 1G 1
  6. P1 1G 2
• Put aside from cultures 6/7 (labelled with this date)
  1. P2 15L 1
  2. P2 15L 2
  3. P4 8J 1
  4. P4 8J 2
  5. P1 5H 1
  6. P1 5H 2

Stored at -80

Liquid Culture
Cultured the following in 5mL LB

• I15010 1 (Kan)
• I15010 2
• P1 11H 1 (Amp)
• P1 11H 2

Also placed Kan plate in the incubator to test antibiotic efficiency - no growth on 8/7

7 July 2007

• Made 10x TAE Buffer

Digest Gel

• Prepared 8 lane 60mL agarose gel with 1xTAE buffer.
• Loaded 20uL of digest samples from 6/7 in the following lane order.
  1. P4 8J 1
  2. P4 8J 2
  3. P1 5H 1
  4. P1 5H 2
  5. P2 15L 1
  6. P2 15L 2

Glycerol Stocks
The following Glycerol Stocks were made and dated 7/7:

• I15010 1
• I15010 2
• P1 11H 1
• P1 11H 2

Stored at -80

Miniprep

• Miniprepped the remains of the cultures and labelled with todays date 7/7. Samples were eluted with TE buffer.

Digest

• Digested 5uL of each of the above miniprep DNA
  • EcoRI/PstI in buffer 3
    • I15010 1 (Kan)
    • I15010 2
    • P1 11H 1 (Amp)
    • P1 11H 2
• Incubated for 3hours at 37degrees
• Added 5uL 6x loading dye and stored at -20

8 July 2007

Transformation
Resuspended and transformed the following

• P1 15P(BBa_R0082, Omp R+, Amp)
• P1 17H(BBa_R0083, truncated BBa_R0082 Omp R+, Amp)
• P1 11A(BBa_E0430, EYFP(RBS+,LVA-,term) Amp)
• P1 16E(BBa_E0430; RBS,GFP,term; Amp)

The following was also retransformed due to colonies on previous plate appearing to be contaminants and failure of plasmid isolation from these colonies.

• P2 13K (BBa_Q04510, c1 inverter, Kan)

Week 3

9 July 2007

• Digested I15010 (E/P), P1 11H (E/H), P2 15L (E/P) 1.5hrs run
• liquid culture P1 11H, I15010, P1 15P, P1 16E, P1 17H, P2 13K

• Loaded and ran Gel:
  1. DNA ladder 1kb+
  2. E/P I15010
  3. E/P I15010
  4. E/H P1 11H
  5. E/H P1 11H
  6. Undigested P2 15L
  7. Undigested P2 15L
  8. E P2 15L
  9. E P2 15L
  10. P P2 15L
  11. P P2 15L
  12. E/P P2 15L
  13. E/P P2 15L
  14. DNA ladder 1kb+

Ran for 1.5 hours

10 July 2007

• Miniprep
• Digested P1 16E, P1 11A, P2 13K, I15010, P1 15P, P1 11H, P1 17H
• liquid culture I15010, P2 13K

• Loaded and ran Gel:
  1. DNA ladder 1kb+
  2. E/P P1 16E
  3. E/P P1 16E
  4. E/P P1 11A
  5. E/P P1 11A
  6. E/P P2 13K
  7. E/P P2 13K
  8. E/P I15010
  9. E/H P1 15P
  10. E/H P1 15P
  11. E/H P1 11H
  12. E/H P1 11H
  13. E/H P1 17H
  14. E/H P1 17H
  15. DNA ladder 1kb+

Ran at 95V for 1 hour

11 July 2007

• Digested for ligation P1 15P(1)10/7, P1 11H 10/7, P1 17H(1)10/7, P1 16E(2)10/7, P1 11A(1)10/7
• Loaded:
  1. X/P P1 11A
  2. X/P P1 16E
  3. S/P P1 15P
  4. S/P P1 11H

• Excise bands of interest and purify using invitrogen kit
• liquid culture P1 11A, P1 15P 10ml
• Transformed P2 21B, P2 23N, P3 20I into DB3.1 heat shock.
• Glycerol Stocks P1 11A, P1 16E, P1 11H, P1 15P, P1 17H

12 July 2007

• Ran Gel:
  1. 20kB+ ladder
  2. P1 11A
  3. P1 16E
  4. P1 15P
  5. P1 11H

(samples from gel purified DNA of 11/7)

• Minipreped P1 11A, P1 15P
• Digest:
  1. S/P+ Alkaline Phosphatase of P1 15P
  2. X/P of P1 11A

-Did not work: too much DNA.

• Ligate control=(2uL ligase buffer,1uL ligase,5uL vector(P1 15P),12uL H20)
• Ligate (2uL ligase buffer,1uL ligase,5uL vector(P1 15P),10uL insert(P1 11A),2uL H20)
• liquid culture transformants 11/7

13 July 2007

• Minipreped cultures from transformants 11/7
• Digested
  1. S/P P1 15P
  2. X/P P1 11A

from 12/7/07 37degC 3hours 15 minutes stopped by 5uL of 6X Loading dye.

• Ran on Gel for 1 hour
• Excise bands 800bp from P1 11A, 2Kbp from P1 15P and purified.
• Glycerol Stocks of P3 20I, P2 21B, P2 23N
• Transform DH5a with ligation product from 12/7.

14 July 2007

• Transformation

-results: 6 colonies on control plate
5 colonies on ligation plate
6 colonies on transformation of digested DNA from Thursday

Week 4

16 July 2007

• Colony PCR of all (5) colonies from ligation plate (14/7) and one from control plate.
• Digested same colonies at the same time to confirm that colony PCR was working.

-results: No colonies were apparent.

17 July 2007

• Set up repeat of Ligation from 12/7.

18 July 2007

• Purchased Restriction enzymes XbaI, EcoRI, PstI
• Miniprepped cultures 1,3,6 from 16/7
• Digested with E/P in triplicate.
• Ran Gel:
  1. DNA ladder,
  2. ctrl (from PCR reaction 1 earlier)
  3. Digest 1 (P1 15P)
  4. Digest 3 (P1 15P)
  5. Digest 6 (P1 15P)
  6. Digest from 12/7 1(P1 11A)
  7. Digest from 12/7 2(P1 11A)

• Transformed
  1. pN26
  2. pN29

with controls: P blank plate, C competent cells not exposed to DNA B LB step on ways.

-observe B+P plates clean. Small colonies on NL29 and NL26 plates as well as C.

• Picked three colonies from each plate and cultured.

20 July 2007

• Transformation of ligations from 17/7 together with a control of undigested P1 15P and competent cells only.

21 July 2007

• Transformation results from 20/7: 0 colonies on competent cells only control, many on undigested vector, and 3 on ligation plate.