From 2007.igem.org

September 2007
Sa	Su	M	Tu	W	Th	F
1	2	3	4	5	6	17
8	9	10	11	12	13	14
15	16	17	18	19	20	21
22	23	24	25	26	27	28
29	30

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Contents 1 September 2007 1.1 September 4 1.2 September 5 1.3 September 7 1.4 September 11 1.5 September 12 1.6 September 13 1.7 September 14 1.8 September 17 1.9 September 19 1.10 September 24 1.11 September 25 1.12 September 26 1.13 September 28

September 2007

September 4

Performed a screening digest of E0434 & I5611 after a midiprep. Results: Not so good, however, linear cuts showed that the DNA was present, so ligation will go ahead.

September 5

• Transformed I5611 & E0434 again in Top 10 cells. Used a 2 min heat shock time, and plated on Amp/Kan (E0434) and Amp (I5611) plates.
• Did a large scale digest I5611 & E0434. However, there might not have been enough BsrGI enzyme added, hence cuts were not clear and pronounced. Digest must be performed again.

September 7

• Ligated linear I5611 & E0434. Left overnight at room temperature instead of at 37C in a water bath.
• Then performed a screening digest of I5611 & E0434. This proved unsuccessful again.

September 11

• Got excellent colonies for all ligation plates (used- Insert:Vector [1:1, 1:2, 1:3, 1:4, 1:5]x2). Each plate had ~25 colonies.
• Seeded 12 colonies from each plate, in 1X LB with Amp/Kan resistance. Thus, seeded 60 colonies in total, the culture tubes were then left in the shaker/incubator at 5.30pm, to be kept overnight.

September 12

-Last night's seedings were ecellent, with clear, visible growth on all culture tubes.

• Performed a miniprep of all the seedings.

-Will perform an overnight screening digest to see if the ligation construct is indeed there.

September 13

Screening digest performed on ligation. -Enzymes used: EcoRI & EcoRV [cuts at 4435bp & 1906bp], Linear: EcoRI [cuts at 6341bp] -Used both a large and small gel, with alternating linear and double cuts for each insert vector ratio colony.

• Results: Inconclusive.

September 14

Due to previous inconclusive results, same screening digest experiment was performed using the same enzymes.
Results: Screening digest again showed bands at ~4kb and at 2kb. This was not expected. However, after looking at the enzymes used again, there is a possibility that the ligation did not occur as similar cuts were found in individual cells.

September 17

Performed another screening digest, this time with enzymes that are more spaced out when producing bands, so that we can identify if the enzymes are cutting or not. -Enzymes used: XmnI & BanII -Cuts: Ligation - [4206, 2096bp], I5611 - [4206, 2096bp], E0434 - [3196, 910bp] -Buffer 4 + BSA.

• Results: Bad gel ruined results

September 19

Screening digest again! This time using StuI & EcoRI. Cuts- 4104bp and 2237bp, using Buffer 2 + BSA.
Results: No construct present in the ligation.

September 24

As the earlier ligation did not work, we did a ligation again, of E0434 to I5611.
did step one of protocol today, performed a large scale digestion using the following recipe: 5ul Buffer 2 5ul 10X BSA 18ul DNA 1ul BsrGI 1ul SpeI

• Then digest overnight @37C.

September 25

• Found that the overnight digestion was left at 30C instead of 37C. Taken out after 18 1/2 hours of incubation.
• Restriction enzymes were deactivated at 80C in a water bath for 20 min.
• Newly cut DNA were kept in the freezer as I (Avi) did not have enough time to perform the screening digest.

September 26

Screening digest performed using BsrGI and SpeI, -For I5611, cuts at 6150bp & 152 bp -For E0434, cuts at 3915bp & 191 bp
Results showed that cuts were successful.

September 28

Screening digest on ligation performed, using both large and small gels, testing on a large quantity of insert:vector colonies, all proved unsuccessful.