Melbourne/Plan:Gas vesicles
From 2007.igem.org
(Difference between revisions)
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## Ncbi genebank AF053765 [[Melbourne/AF053765|(local copy)]] [http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nuccore&id=3089519| (original source)] [[Melbourne/AF053765FASTA| (FASTA seq.)]] | ## Ncbi genebank AF053765 [[Melbourne/AF053765|(local copy)]] [http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nuccore&id=3089519| (original source)] [[Melbourne/AF053765FASTA| (FASTA seq.)]] | ||
## pBluescriptIIKS+ [http://www.stratagene.com/products/displayProduct.aspx?pid=267 (stratagene) ][http://www.stratagene.com/vectors/sequences/pbl2ksp_s.txt (sequence) ] [http://www.stratagene.com/vectors/restriction_sites/pbl2ksp_r.txt (restriction map) ][http://www.stratagene.com/vectors/maps/pdf/pBluescript%20II%20KS+_%20webpg.pdf (map) ] [http://www.stratagene.com/manuals/212205.pdf (Manual)] | ## pBluescriptIIKS+ [http://www.stratagene.com/products/displayProduct.aspx?pid=267 (stratagene) ][http://www.stratagene.com/vectors/sequences/pbl2ksp_s.txt (sequence) ] [http://www.stratagene.com/vectors/restriction_sites/pbl2ksp_r.txt (restriction map) ][http://www.stratagene.com/vectors/maps/pdf/pBluescript%20II%20KS+_%20webpg.pdf (map) ] [http://www.stratagene.com/manuals/212205.pdf (Manual)] | ||
| - | ##*[[Melbourne/pBluescriptIIKS nospaces|(no spaces sequence)]] | + | ##*[[Melbourne/pBluescriptIIKS nospaces|(no spaces sequence 2961bp)]] |
##*[[Melbourne/pBluescriptIIKS PstI HindIII |(no spaces sequence cut at PstI & HinIII)]] | ##*[[Melbourne/pBluescriptIIKS PstI HindIII |(no spaces sequence cut at PstI & HinIII)]] | ||
##*Total 2961 base pairs: | ##*Total 2961 base pairs: | ||
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##*SpeI cuts at 683 | ##*SpeI cuts at 683 | ||
## pNL26 Plasmid with insert:[[Melbourne/cannon plasmid pNL26 seq| (seq)]] [[Melbourne/cannon plasmid pNL26 res map| (res map)]] [[Melbourne/cannon plasmid pNL26 HindIII digest|(HindIII digest)]] | ## pNL26 Plasmid with insert:[[Melbourne/cannon plasmid pNL26 seq| (seq)]] [[Melbourne/cannon plasmid pNL26 res map| (res map)]] [[Melbourne/cannon plasmid pNL26 HindIII digest|(HindIII digest)]] | ||
| - | ## pNL29 Plasmid with insert:[[Melbourne/cannon plasmid pNL29 seq| (seq)]] [[Melbourne/cannon plasmid pNL29 res map| (res map)]] [[Melbourne/cannon plasmid pNL29 HindIII digest| (HindIII digest)]] | + | ## pNL29 Plasmid with insert:[[Melbourne/cannon plasmid pNL29 seq| (seq 6041)]] [[Melbourne/cannon plasmid pNL29 res map| (res map)]] [[Melbourne/cannon plasmid pNL29 HindIII digest| (HindIII digest)]] |
Revision as of 21:57, 12 July 2007
Steps:
- Sequences:
- Ncbi genebank AF053765 (local copy) (original source) (FASTA seq.)
- pBluescriptIIKS+ (stratagene) (sequence) (restriction map) (map) (Manual)
- (no spaces sequence 2961bp)
- (no spaces sequence cut at PstI & HinIII)
- Total 2961 base pairs:
- HindIII cuts at 719
- EcoRI cuts at 707
- PstI cuts at 701
- Xbal cuts at 677
- SpeI cuts at 683
- pNL26 Plasmid with insert: (seq) (res map) (HindIII digest)
- pNL29 Plasmid with insert: (seq 6041) (res map) (HindIII digest)
- Recovery of genes: (2 days)
- Recover the plasmid from paper provided into solution.(method)
- Transform E.Coli strain DH5alpha. Screen with Amp 100ug/ml (as per ref.)(electroporation & heatshock)
- pick 3 colonies of each
- Overnight Culture x9(6 above and 3 form agar block provided)
- Miniprep
- Produce glycerol stocks
- Confirm presence in recovered sample using digest.(HindIII)
- ->Established Supply of Plasmid
- Induce translation overnight 37deg with IPTG in 100ml plates (method)(2days)
- Confirm transcription RT-PRC,
- Confirm translation (buoyant phenotype method).
- Confirm translation (Namarski optics (direct interferance contrast microscopy method).
- Removal of four biobrick like restriction sites all in GvpL.
- EcoRI [GAATTC] in gvpL (2858)
- PstI [CTGCAG] in gvpL x 3 (2522,2900,3005)
- XbaI [TCTAGA] (not present)
- SpeI [ACTAGT] (not present)
- Insertion of biobrick required restriction sites by PCR primer modification.(method)
- Design of primers
- Primer generation
- Plasmid extraction from culture
- PCR
- Restriction EcoR1 & Spe1
- Gel separation
- Restriction of standard Library death plasmid EcoR1,Spe1.
- Ligation.
- Transform host with regulated POPS output
- Confirm dna, rna , protein (as for A)
