Melbourne/Plan:Gas vesicles

From 2007.igem.org

(Difference between revisions)
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##      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 2961bp)]]   
##*[[Melbourne/pBluescriptIIKS nospaces|(no spaces sequence 2961bp)]]   
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##*[[Melbourne/pBluescriptIIKS PstI HindIII |(no spaces sequence cut at PstI & HinIII)]]
+
##*[[Melbourne/pBluescriptIIKS PstI HindIII |(PstI (*TCGAC...CTGCA*) HindIII sequence of cut plasmid 2932bp)]]
##*Total 2961 base pairs:
##*Total 2961 base pairs:
##*HindIII cuts at 719
##*HindIII cuts at 719
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##*Xbal cuts at 677
##*Xbal cuts at 677
##*SpeI cuts at 683
##*SpeI cuts at 683
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##      pNL26 Plasmid with insert:[[Melbourne/cannon plasmid pNL26 seq| (seq 7376)]] [[Melbourne/cannon plasmid pNL26 res map| (res map)]] [[Melbourne/cannon plasmid pNL26 HindIII digest|(HindIII digest)]]
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##      pNL26 Plasmid with insert:[[Melbourne/cannon plasmid pNL26 seq| (seq 7376bp)]] [[Melbourne/cannon plasmid pNL26 res map| (res map)]] [[Melbourne/cannon plasmid pNL26 HindIII digest|(HindIII digest)]]
-
###    pNL26 insert PstI--HindIII:[[Melbourne/pNL26 insert|(no spaces sequence 7376)]]
+
###    pNL26 insert PstI--HindIII:[[Melbourne/pNL26 insert|(no spaces sequence 7376bp)]]
-
##      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)]]
+
##      pNL29 Plasmid with insert:[[Melbourne/cannon plasmid pNL29 seq| (seq 6041bp)]] [[Melbourne/cannon plasmid pNL29 res map| (res map)]] [[Melbourne/cannon plasmid pNL29 HindIII digest| (HindIII digest)]]
-
###    pNL29 insert PstI--HindIII:[[Melbourne/pNL29 insert|(no spaces sequence 6041)]]
+
###    pNL29 insert PstI--HindIII:[[Melbourne/pNL29 insert|(no spaces sequence 6041bp)]]
# Recovery of genes: (2 days)
# Recovery of genes: (2 days)

Revision as of 22:20, 12 July 2007

Steps:

  1. Sequences:
    1. Ncbi genebank AF053765 (local copy) (original source) (FASTA seq.)
    2. pBluescriptIIKS+ (stratagene) (sequence) (restriction map) (map) (Manual)
    3. pNL26 Plasmid with insert: (seq 7376bp) (res map) (HindIII digest)
      1. pNL26 insert PstI--HindIII:(no spaces sequence 7376bp)
    4. pNL29 Plasmid with insert: (seq 6041bp) (res map) (HindIII digest)
      1. pNL29 insert PstI--HindIII:(no spaces sequence 6041bp)
  1. Recovery of genes: (2 days)
    1. Recover the plasmid from paper provided into solution.(method)
    2. Transform E.Coli strain DH5alpha. Screen with Amp 100ug/ml (as per ref.)(electroporation & heatshock)
    3. pick 3 colonies of each
    4. Overnight Culture x9(6 above and 3 form agar block provided)
    5. Miniprep
    6. Produce glycerol stocks
    7. Confirm presence in recovered sample using digest.(HindIII)
    8. ->Established Supply of Plasmid
  1. Induce translation overnight 37deg with IPTG in 100ml plates (method)(2days)
    1. Confirm transcription RT-PRC,
    2. Confirm translation (buoyant phenotype method).
    3. Confirm translation (Namarski optics (direct interferance contrast microscopy method).
  1. Removal of four biobrick like restriction sites all in GvpL.
    1. EcoRI [GAATTC] in gvpL (2858)
    2. PstI [CTGCAG] in gvpL x 3 (2522,2900,3005)
    3. XbaI [TCTAGA] (not present)
    4. SpeI [ACTAGT] (not present)
  1. Insertion of biobrick required restriction sites by PCR primer modification.(method)
    1. Design of primers
    2. Primer generation
    3. Plasmid extraction from culture
    4. PCR
    5. Restriction EcoR1 & Spe1
    6. Gel separation
    7. Restriction of standard Library death plasmid EcoR1,Spe1.
    8. Ligation.
    9. Transform host with regulated POPS output
    10. Confirm dna, rna , protein (as for A)
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