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Cloning Strategy for IgM heavy and light chains (outdated?)

Order G blocks "mIgM heavy chain against beta amyloid" and "mIgM kappa chain against beta amyloid" that are flanked with complementary sequences to Nelson's primer with Q1 and Qx sites

...

Transform bacteria again?

 

AS-X127 pGGDonr-ENTR-L1-LacZa-L2 has kanamycin resistance.

PCRs to do:

ProductStatusGeneStarting PlasmidStarting Plasmid ResistanceForward PrimerReverse Primer

Tm

hi/lo (anneal)

PCR's that can go together
CD79AcompleteCD79ApENTR CD79A (pDONR223)spectinomyciniGEM KRB 003 FiGEM KRB 004 R62/61 (61)1
CD79A-TCScompleteCD79ApENTR CD79A (pDONR223)spectinomyciniGEM KRB 003 FiGEM KRB 005 R62/61 (61)1
CD79Bnew primers orderedCD79BpENTR CD79B (pDONR223)spectinomyciniGEM_AS001 CD79B Q1_FiGEM_AS002 CD79B QX_stop_R72/70 (70)4
CD79B-TCSnew primers orderedCD79BpENTR CD79B (pDONR223)spectinomyciniGEM_AS001 CD79B Q1_FiGEM_AS003 CD79B Qlinker_R72/70 (70)4
TCS-Gal4VP16completeGal4VP16pEXPR 5 – hEF1a-FGFR-mKate-TCS(L)-G4ampicilliniGEM KRB 001 FiGEM KRB 002 R62/60 (63)6
Lynneed to redoLynpENTR Lyn (pDONR223)spectinomyciniGEM CR 006 FiGEM CR 007 R61/60 (60)2
Lyn-mKatecompleteLynpENTR Lyn (pDONR223)spectinomyciniGEM CR 006 FiGEM KRB 011 R61/60 (60)2
Lyn-(15aa)completeLynpENTR Lyn (pDONR223)spectinomyciniGEM CR 006 FiGEM_AS009 Lyn Qlonglink_R61/60 (60)2
mKateDpnI digest completemKatepENTR_L1_mKate_L2kanamyciniGEM KRB 006 FiGEM KRB 007 R58/61 (58)1
SykcompleteSykpENTR Syk (pDONR223)spectinomyciniGEM_AS004 Syk Q1_FiGEM_AS005 Syk QX_stop_R54/54 (54)3
Syk-eYFPcompleteSykpENTR Syk (pDONR223)spectinomyciniGEM_AS004 Syk Q1_FiGEM KRB 010 R54/54 (54)3
Syk-(15aa)need to redoSykpENTR Syk (pDONR223)spectinomyciniGEM_AS006 Syk Q1_FiGEM_AS007 Syk Qlonglink_R61/59 (59)2
eYFPDpnI digest completeeYFPpENTR_L1_EYFP_L2kanamyciniGEM KRB 008 FiGEM KRB 009 R59/57 (57)5
(15aa)-TEVpneeds DpnI digestTEVppENTR_L1_Tev2kanamyciniGEM_AS010 TEVp Qlonglink_FiGEM_AS011 TEVp QX_R60/60 (60)2

 

Golden Gates:

ProductStatusDNAamountswater
pENTER CD79A+stopIn Progress

CD79A+STOP

.91712.283
pENTR CD79A-TCS-Gal4VP16In Progress

CD79A-TCS

TCS-Gal4VP16

.933

2.262

10.005
pENTER Syk+STOPIn ProgressSyk+STOP1.02912.171
pENTR CD79B-TCS-Gal4VP16Waiting on parts

TCS- Gal4VP16

CD79B-TCS.

  
pENTR CD79B+STOPWaiting on partsCD79B+STOP  
pENTR Gmab HeavyWaiting on partsGmab Heavy  
pENTR Gmab LightWaiting on partsGmab Light  
pENTR Syk-eYFPWaiting on parts

...eYFP

Syk...

  
pENTR Syk-TEVWaiting on parts

Syk...

...TEV

  
pENTR Lyn-TEVWaiting on parts

...TEV

Lyn...

  
pENTR Lyn+STOPWaiting on partsLyn+STOP  
pENTR Lyn-mKateWaiting on parts

Lyn...

...mKate