SBB09Ntbk-Madhvi Venkatesh: Difference between revisions
Line 51: | Line 51: | ||
Based on this, I am sending M10000-1, M10001-2, M10002-1B, M10003-2B for sequencing. | Based on this, I am sending M10000-1, M10001-2, M10002-1B, M10003-2B for sequencing. | ||
==[[User:Madhvi Venkatesh|Madhvi Venkatesh]] 14:46, 2 March 2009 (EST)== | |||
I got my sequencing data back and the results are up on my anderson lab sequencing page. M10000 and M10001 sequenced correctly and these clones are good to use. The wobble products, M10002 and M10003 did not sequence so well. The read for M10002 had Ptet, BBb scar and another short sequence in between the BglII and BamHI sites. The read for M10003 didn't have BglII or BamHI sites and just went from the EcpRI site to the part of the vector after the BamHI site. I double checked the protocols and it looks like I did everything correctly, so I am not sure what has gone wrong. I am going to do a few things: | |||
* send the other clone of M10003 for sequencing hoping for better results | |||
* pick a few extra colonies of M10002 (I am not sure if they are right because I didn't see these guys before and the plate has been sitting out on the bench for a few days) | |||
* set up the wobble reaction and go through the whole process one last time |
Revision as of 12:46, 2 March 2009
Madhvi Venkatesh 14:54, 2 February 2009 (EST)
Today, I got my project ball and I am making a new flagellar display system. I need to consider several possibilities and figure out what would be best to make and the source. The project description if on this page: new flagellar display system
Madhvi Venkatesh 15:40, 9 February 2009 (EST)
Possible places to find EtpA homolog (from CTEC):
APEC 01:k1 CFT073 EHEC 0157:H7 Shigella flexneri
Madhvi Venkatesh 15:24, 23 February 2009 (EST)
So I rewrote my construction files after I realized that I can't display a protein on the N and C terminal ends of fliD because they are not facing the surface. The new versions of the construction files including Chris's notes on them are linked from the construction files page. We got oligos last Wednesday and I set up PCRs for the N and C terminal parts and wobble PCRs for the linkers. I purified and digested over the weekend. I also set up the SOEing PCR at the end of last week. Today I ligated the parts into pBca1256 and transformed. I will pick colonies tomorrow and send for sequencing on Wednesday. I also purified and digested the SOEing PCr for removing the restriction site in the N terminal fliD part.
Here are gel pics from the PCRs:
Here is the result of the SOEing PCR for the M1000 part {<N.fliD!} with the restriction site removed:
Here are the results of the other PCRs:
Madhvi Venkatesh 21:23, 27 February 2009 (EST)
I digestion mapped my basic parts:
Lane | Plasmid | Clone | Enzyme 1 | Enzyme 2 | Expected Bands | Result |
1 | pBca1256-M10000 | 1 | EcoRI | XhoI | 986 + 2113 | correct |
2 | pBca1256-M10000 | 2 | EcoRI | XhoI | 986 + 2113 | correct |
3 | pBca1256-M10001 | 2 | EcoRI | XhoI | 986 + 2248 | correct (not completely digested) |
4 | pBca1256-M10002 | 2 | EcoRI | XhoI | 986 + 1585 | No DNA |
5 | pBca1256-M10002 | 1B | EcoRI | XhoI | 986 + 1585 | correct (not completely digested) |
6 | pBca1256-M10003 | 1B | EcoRI | XhoI | 986 + 1582 | correct (mostly undigested) |
7 | pBca1256-M10003 | 2B | EcoRI | XhoI | 986 + 1582 | correct |
Based on this, I am sending M10000-1, M10001-2, M10002-1B, M10003-2B for sequencing.
Madhvi Venkatesh 14:46, 2 March 2009 (EST)
I got my sequencing data back and the results are up on my anderson lab sequencing page. M10000 and M10001 sequenced correctly and these clones are good to use. The wobble products, M10002 and M10003 did not sequence so well. The read for M10002 had Ptet, BBb scar and another short sequence in between the BglII and BamHI sites. The read for M10003 didn't have BglII or BamHI sites and just went from the EcpRI site to the part of the vector after the BamHI site. I double checked the protocols and it looks like I did everything correctly, so I am not sure what has gone wrong. I am going to do a few things:
- send the other clone of M10003 for sequencing hoping for better results
- pick a few extra colonies of M10002 (I am not sure if they are right because I didn't see these guys before and the plate has been sitting out on the bench for a few days)
- set up the wobble reaction and go through the whole process one last time