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From 2010.igem.org

BRIDGE

5/7/2010

• sacB digest with EcoRI -band around kb (gel 1)

6/7/2010

• sacB digest with EcoRi/PstI. Band also around 5 kb.

(gel 2).

7/7/2010

• PCR of sacB to check insert. Used sacB tube 1 and 3.
• Restriction digest of PCR with EcoRI and NotI.
• (gel no 3)
• lane 1- marker
• lane 2, 3, 4 - sacB 1, 2 & 3 cut with NotI; band size: slightly bigger than 4 kb
• Lane 5- PCR of LuxAB; band size around 1.5. kb
• lane 6- Vector of sacB (pSB1C3, 2072bp, aroun 3kb with RFP) cut with NotI; band size around 1.5. kb
• Lane 7- PCR of sacB with gene sepcific primers; band size around 1.5. kb

• Trouble shooting: sacB cut with NotI give about 4kb, whereas cutting with EcoRI gives 5.5. kb... hmhmhm.

there should be 2 NotI sites on either side of the insert but we only get 1 band--> is there only 1 NotI site? Furthermore, PstI/EcoRI digests give only 1 band (same as cut only with EcoRI), so maybe there is no PstI/NotI site?

• Purifying sacB from PCR product to start making BRIDGE- row F in the BOX 1.

9/7/2010

• Making working solutions of primers from stock: 4ul of H20; 1 ul of stock solution
• Primers: forward 72.3 C, revers- 77.6 C; length of the product: 2.6-2.7 kb

to do on Monday:

• redo sacB- catR ligation
• Run 5 ul of ligation on gel to check for large bands
• redo RLS part transformations

12/7/2010

• Transformants of luxAB-edi1 failed- probably smth wrong at the purification stage...
• (gel no 4)

Lane 1	Lane 2	Lane 3	Lane 4	Lane 5	Lane 6
ladder	luxAB-ediI digest	luxAB-ediI ligation	sacB digest	catR digest	sacB- catR lgation
expec. no of bands	2	1 or 3	1	1	1 band= to 4+5

• If all failed- purification procedure failed
• If 3 and 6 failed- ligation failed
• If all fine then PCR/transformation failed

conclusion:...

• 3 and 6 failed- ligation problem (not enough DNA, ligase).
• Band 1- luxAB but no ediI (?)

• Repeated digest (sacB with XbaI, catR with SpeI, LuxAB & EdiI with SpeI & SacI) and purification.
• Set up ligations with 2ul ligase instead of 1 ul (1ul of H2O less). If this fails- perhaps only 2-3 h ligation ina RT (SpeI is set up in both digest- could be common cause)

13/7/2010

• First, run PCR of catR-sacB ligation (use DNA?????' MK1- with sacBr1 (19.6.10); MK2- sacBr1(10)
• Gel electrophoresis of: digested catR, digested sacB, ligated sacb-catR, digested LuxAB-ediI and ligated luxAB-ediI along with sacB-catR PCR

'(gel no 5'

Lane 1	Lane 2	Lane 3	Lane 4	Lane 5	Lane 6	Lane 7
ladder	ediI	digested LuxAB-ediI	ligated luxAB-ediI	ligated catR & sacB	PCR 1 (MK1)	PCR 2 (MK2)

'AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAARGH!!!!!!!!!!!!!!!1'

• Almost completely convinced it's the ligase... or me (MK). Officially taking myself off the project. xx
• (Spotted 2 purified sacBs in the freezer... maybe try the original if this one fails...?)

'9/8/2010'

Gel with :

• cat + sacB (PCR product) (6)
• vector + cat + sacB (7) (both done by CF)

20/8/2010

• Made plates variety of sucrose combinations--> 40 cml
• 0%
• 0.1%
• 0.25%
• 0.5%
• 1.0%

• Plated E6 miniprep, which should show some sucrose sensitivity (1plate at each concentration)
• Control- E5, 1 plate at each concentration

30/8/2010

• Primers have arrived for up and downstream sequences of TnaA region.

13/09/2010 Plane for week begininng 13th:

• Amplify RFP with primers --> RBS-F2; YFP - R2 ---> to amplify RFP
• Digest UP with SpeI and B-D with XbaI
• Ligate UP to BRIDGE-DOWN
• Digest RFP with XbaI and SpeI
• Ligate UP and RFP
• Ligate DOWN and RFP

• Also- registry editing for RLS (definately dodgy sequence), LovTap
• Justification of reseubmission
• Look up absorption/emission rates and transmission through medium for Donal

20/9/2010

• Achieved: cat/sacB- traA down ligation
• Attempted: traA up- cat/sacB/down ligation; traA up- RFP ligation; RFP- traA down ligation/ ALL FAILED
• Running: purified digest of:

- cat/sacB + SpeI
- tnaA down- XbaI
- tnaA up + SpeI
- cat/sacB/down + XbaI
- RFP + XbaI
and also running: UP- BRIDGE- DOWN ligation; UP - RFP ligation

Have: PCR of cat/sacB, RFP, BRIDGE-DOWN, traA up, traA down; ligation of BRIDGE-DOWN

gel no 7

Run gel:

Lane 1	Lane 2	Lane 3	Lane 4	Lane 5	Lane 6	Lane 7	Lane 8
Ladder	Pure SacB	Pure SacB & EcoRI	sacB & catR PCR	I15009 digest	S284T PCR Product	Purified 356K	Purified 356R

25/10/10
Repetitive failure of purifications with both traditional method and kits, unsure what is going wrong. Have decided to tell Chris. Off to Firbush on Wednesday.

01/10/10
In my absence (at Firbush with the rest of the Biotechnologists) Chris has managed to construct the tnaA UP-cat-sacB-tnaA DOWN segment that we need for the first part of the BRIDGE protocol. I will continue to build the tnaA UP-RFP-tnaA DOWN segment.
Set up ligations of RFP-DOWN and UP-RFP, left in 16C incubator overnight.

02/10/10
Lab on a Saturday again, fun fun fun...
Retrieved and amplified ligations and ran on gel. UP-RFP appears to be significantly shorter than RFP-DOWN and indeed shorter than it ought to be (should be about 2kb, is only around 1.5kb).
Purified both products, then digested RFP-DOWN with XbaI. (Used 2hour incubation time to write presentation for Wednesday).
Set up ligation of UP with RFP-DOWN and left in incubator overnight.

03/10/10
Bus to Darwin for 11:20am; enter Darwin; lift to 8th floor; retrieve ligations from waterbath in cold room; walk down to 7th floor; put ligation in freezer; lift to ground floor; leave Darwin; catch 11:40am bus back to central.

04/10/10
PCR of ligation of UP-RFP-DOWN. Made and ran gel along with purified PCRs from Saturday, just in case they're still not working.
They worked! The purifications were perfect. There is a band about the size of UP-RFP-DOWN in it's lane, but it's quite faint. I will probably repeat the PCR with a lower annealing temperature (around 50) this afternoon. I suspect the UP primers do not anneal properly above 54C.

07/10/10
Chris set up overnight cultures of DH5-alpha cells and JM109 cells, both containing the lambda-red plasmid, in 2.5ml LB with tetracycline.

08/10/10
Took 2 5ml LB bottles:
- Added 1.7 microlitres of tet15 and 0.3ml DH5-alpha overnight culture to one
- Added 5 microlitres of tet15 and 0.3ml of JM109 overnight culture to the other

Grew second cultures for 2 hours at 30C with shaking
Measured optical density after 2 hours:
- Water = 0.00
- DH5-alpha = 0.251
- JM109 = 0.320

Added 100microlitres arabinose to both cultures and grew at 37C for one hour with shaking.
Electroporated cells and transformed with tnaAup-cat-sacB-tnaAdown construct