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From 2014.igem.org

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NOTEBOOK ENTRIES

6/19:

made high school presentations
re-cultured Syk and Lyn
designed Lyn-TEVp fusion

-Syk and Lyn concentrations found to be only ~15 ng/uL, so need to grow new cultures and re-miniprep
-Primers iGEM2014-EE-[003,006] found to be missing spacer before BsaI site; corrected primers re-ordered


6/20:

miniprepped Syk/Lyn
NEGEM


6/22:

PCRs
-CD79A-stop
-CD79A-TCS
-mKate
sequenced Syk/Lyn


6/23:

PCR purifications from 6/22
imaged gels from 6/22
PCRs
-CD79B-stop
-CD79B-TCS
-TCS-Gal4


6/24:

DpnI digest for mKate and eYFP
purified and verified PCR for Syk-stop, eYFP


6/25:

redid Syk-eYFP PCR using DMSO
imaged gels for yesterday's PCRs
purified, ran gel for Syk-eYFP PCR
ran remaining PCRs
-Lyn-stop
-Lyn-mKate
-Lyn-(15aa)
-Syk-eYFP
-Syk-(15aa)
-(15aa)-TEVp
purified, gelled remaining PCRs
uploaded gel images to parts pages
Golden Gates for CD79A, CD79A-TCS-Gal4, Syk

-all of the originally ordered primers have arrived
-CD79B PCRs failed again, even with DMSO
-new CD79B primers designed/ordered
-when loading the gel, the Lyn-stop wouldn't fill the wells, but instead seemed to float up out of the gel on top of the TAE - ?


6/26:

redid Syk-15aa and Lyn PCRs using DMSO
redid CD79B PCRs using betaine
purified, gelled PCR products
ran DpnI digest on TEVp product
transformed/plated Golden Gates for CD79A, CD79A-TCS-Gal4, Syk
Golden Gates for Lyn, Lyn-mKate, Lyn-TEVp, Syk-TEVp, Syk-eYFP


6/27:

picked colonies for CD79A, CD79A-TCS-Gal4, Syk golden gates
transformed/plated golden gates for Lyn, Lyn-mKate, Lyn-TEVp, Syk-TEVp, Syk-eYFP
PCRed Gmab heavy and light chains
re-Golden Gated CD79A, CD79A-TCS-Gal4, Syk
Golden Gated Gmab heavy/light chains
designed sequencing primers
pUC19 competence


6/28:

miniprepped CD79A, CD79A-TCS-Gal4, Syk
transformed/plated repeat CD79A, CD79A-TCS-Gal4, Syk
transformed/plated Gmabs


6/29:

picked colonies for Lyn, Lyn-mKate, Lyn-TEVp, Syk-TEVp, Syk-eYFP
picked colonies for repeats
picked colonies for Gmabs


6/30:

miniprepped Lyn, Lyn-mKate, Lyn-TEVp, Syk-TEVp, Syk-eYFP


7/1:

digested and gelled pENTR Syk, Syk-eYFP, Syk-TEVp, Lyn, Lyn-mKate, Lyn-TEVp, CD79A, CD79A-TCS-Gal4
-Lyns and Syk-eYFP failed
PCRed CD79Bs using new primers; Gmab H and L gBlocks
ran gel extraction on Gmab H and L PCR products
digest-verified pENTR CD79A, CD79A-TCS-Gal4, Syk, Syk-TEVp


7/3:

digested and gelled redone Syk-eYFP and Lyns
-failed again


7/7:

digested and gelled pENTR Lyn, Lyn-mKate, Lyn-TEVp, Syk-eYFP, CD79B, CD79B-TCS-Gal4, Gmab H, Gmab L, pEXPR CD79A, CD79A-TCS-Gal4, Syk, Syk-TEVp
-CD79B, Lyn, Syk-eYFP questionable; Lyn-TEVp, Lyn-mKate failed
digest-verified pENTR CD79B, CD79B-TCS-Gal4, Gmab H, Gmab L; pEXPR Hef1a:CD79A, Hef1a:CD79A-TCS-Gal4, TRE:Syk, Tre:Syk-TEVp


7/9:

sequenced pENTRs for Lyn, Lyn-mKate, Lyn-TEVp, Syk, Syk-eYFP, Syk-TEVp, CD79A, CD79B, CD79A-TCS-Gal4, CD79B-TCS-Gal4, Gmab L, Gmab H


7/11:

ran PCR screen for Syk-eYFP, Lyn, Lyn-mKate, Lyn-eYFP
-all the Syk-eYFPs failed; each of the Lyns had a few that might have worked
ran gels for CD79B-TCS-Gal4


7/12:

ran screening gel for CD79B
-some seemed to work


7/16:

digest-verified pEXPR Hef1a:Gmab L, Hef1a:Gmab H


7/17:

ran gels for pENTR Syk-eYFP, pEXPR Hef1a:CD79B-TCS-Gal4
-Syk-eYFP failed


7/21:

ran gels for pENTR Lyn, Lyn-TEVp, Lyn-mKate; pEXPR CD79B, CD79B-TCS-Gal4
-seemed to have some that worked for each


7/27:

Western blot for Syk (first try)
-inconclusive since errors in setup, protocol, etc.; no bands


7/29:

digested and gelled pENTR Syk-eYFP, pEXPR Lyn-mKate
-Syk-eYFP failed, but Lyn-mKate worked


7/30:

digested and gelled pEXPR Lyn-TEVp
-all failed


8/1:

Syk western blot (second try)
-successful this time
-results: Ramos and HEK293 cells both have high levels of endogenous Syk.

BCR membrane localization experiment (second try)
-this time, had B cells to use as positive control (also used this control data for next experiment)
-bleedthrough problems in cytometry data, but seemed to be some visible localization


8/2:

ran gels for pEXPR Lyn-TEVp, Syk-eYFP
-Syk-eYFP seemed to work, but Lyn-TEVp didn’t


8/7:

BCR membrane localization experiment (third try)
-blind transfection to avoid bleedthrough problems
-showed that there was localization, but only a small amount (~1.7%, as opposed to ~95% in B cells)


8/11:

ran gels for pEXPR Lyn, Lyn-TEVp, TRE:Syk-eYFP
-seemed to fail


8/14:

ran gels for Gibsons, pEXPR Gmab L, CD79A, CD79B
-Gibsons failed, probably since grown up at 37 degrees instead of 30; other plasmids worked


8/20:

digested and gelled redone Gibsons


8/21:

digested and gelled Gibsons, pEXPR TRE:CD79A, TRE:CD79B, TRE:Gmab L, TRE:Gmab H, TRE:Lyn-mKate, Hef1a:Lyn
-A4/B4 Gibson passed, as well as the other constructs