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What to do when you generate a new plasmid

1) Give it a unique plasmid name, such as pLJ# (p stands for plasmids, and LJ is the initial. # should be in numeric order), use your own initials for the plasmids you made.

2) Write details about how you create this plasmid

  • a. Include primers and template you used.
  • b. Write down the vector backbone.
  • c. Indicate whether it has been sequenced (which you always should) and a summary of sequencing result (such as whether it is 100% wt, or contains silent mutations). Indicate where the sequencing result is stored.

3) Enter into the database on wiki and indicate the antibiotic resistance.

4) Draw a plasmid map using SNAPgene software

Example: you generate a plasmid which is lite-1p::YFP
Example of details: Primer pair xxx and xxx are used to amplify lite-1 promoter from N2 genomic DNA (3kb). Primer pair xxx and xxx are used to amplify YFP from plasmid pMS1 (0.7kb). They are cloned into the vector xxx through Gibson Assembly. Primers xxx are used for sequencing. lite-1 promoter region is 100% correct, while there is a silent mutation at position xxx for YFP. Sequencing result can be found at xxx.

Lab Plasmids Database

plasmid # Name Description Primer used Status Where Source insert size Comments Backbone Backbone size Frozen Ref
pMS1 odr-1p::cGAL-N::let-858 3'UTR split gal-4 cGAL-N expressed in gcy-10(odr-1) promoter expressing in I1 and others (AWC, AWB). DBD derived from pHW530 1.8kb 3.7kb
pMS2 ehs-1p::cGAL-C::let-858 3'UTR split gal-4 cGAL-C expressed pan-pharyngeal neurons. AD derived from pHW522 62bp might be issues with the ehs-1 promoter 3.3kb
pMS3 ehs-1p::his24::wrmScarlett should be red in all pharyngeal neurons. derived from PJ20 Anuj no expression when injected
pMS4 ehs-1p::his24::GCaMP7s should be GCaMP in all pharyngeal neurons. derived from PJ20 Anuj
pMS5 pHW522 Prab-3::NLS::gp41-1-C-intein::cGAL(AD)::let858 3'UTR NA 3.3kb
pMS6 pHW530 Prab-3::NLS::cGAL(DBD)::gp41-1-N-intein::let-858 3′UTR NA 3.7kb
pMS7 pHW393 Prab-3::NLS::cGAL(DBD):: cGAL(AD)::let-858 3′UTR 1.2kb 3.6kb
pMS8 pBX (pha-1 rescue)
pMS9 rab-3P::2NLS::GFP(65C)
pMS10 pAS1-his-24::GCaMP7s
pMS11 pAS1-his-24::tagBFP should be rab-3 promoter
pMS12 pAS1-his-24::tagRFP should be rab-3 promoter
pMS13 pAS1-his-24::mNeptune should be rab-3 promoter
pMS14 pAS1-his-24::mCherry should be rab-3 promoter Addgene 124348
pMS15 pAS1-his-24::linker::wrmScarlet should be rab-3 promoter
pMS16 pAS1-his-24::CyOFP::egl-13NLS should be rab-3 promoter
pMS17 pPHA2∷GFP-F GFP expressed in I4 from Marc Pilon pPD95.69
New plasmids
pLJ1 Pceh-28::NLS::cGAL(DBD):: cGAL(AD)::let-858 3′UTR oLJ17/18 sequenced Jun 2.5kb M4 specific pHW393 3.6kb yes PMID: 20713707 (primer sequence not provided)
pLJ2 Podr-1 long::NLS::cGAL(DBD):: cGAL(AD)::let-858 3′UTR oLJ27/28 sequenced Jun 1.7kb I1, AWB, AWC pHW393 3.6kb yes primer sequence provided by Nikhil Bhatla, who says they are from PMID: 16547101
pLJ3 Podr-1 short::NLS::cGAL(DBD):: cGAL(AD)::let-858 3′UTR oLJ27/29 sequenced Jun 0.7kb putative I1 pHW393 3.6kb yes Educated guess, based on information from Jihye Yeon, Sengupta Lab
pLJ4 Pceh-34::NLS::cGAL(DBD):: cGAL(AD)::let-858 3′UTR oLJ25/26 sequenced Jun 3.6kb pan pharyngeal neruons pHW393 3.6kb yes PMID: 19879847
pLJ5 Pceh-2::NLS::cGAL(DBD):: cGAL(AD)::let-858 3′UTR oLJ45/46 sequenced Jun 1.2kb I2, I3, NSM pHW393 3.6kb yes NeuroPAL
pLJ6 Ptph-1::NLS::cGAL(DBD):: cGAL(AD)::let-858 3′UTR oLJ47/48 sequenced Jun 0.2kb NSM specific pHW393 3.6kb yes PMID: 30580965
pLJ7 Ptrxr-1::NLS::cGAL(DBD):: cGAL(AD)::let-858 3′UTR oLJ39/40 sequenced Jun 0.8kb M2 specific pHW393 3.6kb PMID: 22836943 (primer sequence not provided)
pLJ8 Plin-44::NLS-tagBFP::let-858 3'UTR oLJ51/52 for Plin-44 and oLJ53/54 for NLS-tagBFP sequenced 2.6kb for Plin-44 and 0.9kb for NLS-BFP pHW393 without GAL4 (FseI+KpnI) 2.7kb yes PMID: 32188430
pLJ9 Pins-10::NLS::cGAL(DBD):: cGAL(AD)::let-858 3′UTR oLJ43/44 sequenced 5.4kb pHW393 3.6kb yes PMID: 24806678
pLJ10 Podr-1 long::NLS::gp41-1-C-intein-cGAL(AD)::let-858 3′UTR subcloned from pLJ2 (odr-1p) and pJL081 (backbone, similar to pHW522) sequenced 1.7kb for split GAL4, C-term equivalent to pHW522 3.3kb No.
pLJ11 Pceh-34::NLS::cGAL(DBD)::gp41-1-N-intein::let-858 3′UTR subcloned from pLJ4 (ceh-34p) and pJL080 (backbone, similar to pHW530)

Gift Plasmids Database

Plasmid name Description Notes antibiotics Source Plasmid location -80
pMS17 pPHA2∷GFP-F GFP expressed in I4 amp Marc Pilon. They did not give it a name, and suggested us to name it by ourselves, so this plasmid appears in both this and our lab databases. B4 yes
pCFJ90 Pmyo-2::mCherry::unc-54utr amp A1 yes
pGH8 pRAB-3::mCherry::unc-54utr do not use. amp very likely wrong after sequencing B5, B6
Addgene_104483 pGP-CMV-jGCaMP7f Kan C1, C2 yes
pPD96.48 myo-2 promoter + unc-54 3’utr cloning vector with myo-2 promoter Amp Gift from Ian Chin-Sang A2 yes
pPD95.75 promoterless + NLS-GFP, unc-54 3'utr Amp Gift from Ian Chin-Sang A3 yes
pBSII SK+ cloning vector Amp Gift from Ian Chin-Sang A5 yes
odr-1p::rfp odr-1 promoter driving rfp, co-injection marker Amp Gift from Ian Chin-Sang A4 yes
pJLR11 [tph-1(short NSM fragment)::del-7-sl2-GFP] Amp Gift from Steven Flavell B3 yes
pLJ080 unc-17p sGAL4sk gp41 N intein Amp Gift from Paul Sternberg A6 yes
pJL081 ceh-19bp sGAL4sk gp41 C-intein.ape Amp Gift from Paul Sternberg A7 yes
pHW530 Prab-3::cGAL-N::let-858 3'UTR Amp Gift from Paul Sternberg A8 yes
KP1368 Pmyo-2::nls::mCherry co-injection marker Amp Gift from Paul Sternberg A9 yes
pENTR-lin-44p pENTR-lin-44 Kan Gift from Yuichi Iino B1 yes
ser-2p NLS-tagBFP pG-ser2(prom2)p_svNLS2-TagBFPsyn Amp Gift from Yuichi Iino B2 yes
wiki/documentation/plasmids.txt · Last modified: 2020/09/24 05:33 by jliu