IGEM:Imperial/2010/PyrD Vector

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Overveiw of PyrD assembly
Overveiw of PyrD assembly

Contents

Week 6

Week 6 Monday Tuesday Wednesday Thursday Friday
Morning
  • Restriction digest of 5' dif XP with XbaI and PstI
Afternoon
    Start assembly of PyrD vector
  • Overnight annealing of 5' Ins ( synthesized oligos )
  • Gel analysis of resultant products from 5' dif XP digest
  • PCR purification of cut 5' dif XP

Thursday, August 12
  • Annealing the forward and reverse strands of the dif XP oligo

The forward and reverse strands of the 5' dif site with XbaI and PstI restriction sites on either side have been synthesized separately. The synthesized fragments arrive in solid powder form. These were immediately diluted in ddH2O to obtain a stock concentration of 1 ng/ul. They were then allowed to anneal together by first heating them to 95 degs for denaturation and allowing them to cool down and anneal overnight.

Friday, August 13
  • Restriction digest of dif XP

After annealing the two strands, the oligo was cut with XbaI and PstI to obtain overhangs that would later ligate with the compatible overhangs of a cut vector.

  • PCR purification of the cut dif XP

The cut oligo was PCR purified in order to get rid of any contaminants. PCR purification gets rid of short pieces of DNA which are less than about 40 base pairs.

  • Gel Analysis of dif XP

Week 7

Week 7 Monday Tuesday Wednesday Thursday Friday
Morning
  • Restriction digestion of 5' ins [K143008] (using Eco and Spe)
  • PCR amplification of vector backbone pSB1C3
  • Gel analysis and extraction of 5' ins
  • Gel purification of 5' ins
  • Restriction digestion of pSB1C3 (using Eco and Pst)
  • PCR purification of pSB1C3 after digestion
  • Gel analysis of 5' ins, dif and pSB1C3 to work out ratios for ligation
  • Restriction digestion of pveg promoter and RBS [K143053] (using Eco and Spe)
  • Restriction digetion of SpecR-T [K143065] (using Xba and Pst)
  • Replica plating and colony PCR of transformed colonies (containing 5' dif in pSB1C3)
  • Gel extraction and PCR purification of pveg and SpecR-T in preparation for ligations this afternoon
  • Gel analysis of pveg , Spec-T and pSB1C3 in order to work out ratios for the ligation
  • Transformation of overnight ligations: 5' ins, dif with pSB1C3 and pveg, SpecR-T and pSB1C3
  • Replica plating and colony PCR of: 5' ins, dif with pSB1C3 and pveg, SpecR-T and pSB1C3
Afternoon
  • PCR purification of PSB1C3 vector
  • Ligation of 5' ins and dif with pSB1C3 - a bench ligation (1 hour) and an overnight ligation were set up
  • Transformation of E.Coli with the bench ligated products
  • Gel analysis and extraction of pveg and SpecR-T
  • Overnight ligation of pveg and SpecR-T with pSB1C3
  • Gel analysis of colony PCR products from the transformations
  • Overnight annealing of diff PES (Synthesized oligo)


Monday, August 16
  • Restriction digest of 5' ins [K143008]

K143008 is the 5' integration site for the PyrD vector. This will be used as a front insert together with the dif XP for the pSB1C3 vector.

  • PCR amplification of pSB1C3

The pSB1C3 vector backbone from the registry was amplified with the use of SB3 and SB2a primers. Submission of parts to the registry requires them to be in a pSB1C3 vector therefore any parts to be submitted will be inserted into this vector.

  • PCR purification of pSB1C3

The PCR amplified vector was purified in order to get rid of any contaminants. For example, short pieces of DNA like the primers.

Tuesday, August 17
  • Gel extraction and purification of 5' ins ES

The digested 5' ins was first analyzed on the gel to verify it's size and then extracted for purification. The 5' ins was gel purified in order to extract only the relevant piece of DNA.

  • Restriction digest of pSB1C3

The pSB1C3 vector was digested so that it would contain compatible overhangs for ligation with inserts.

  • PCR purification of pSB1C3 EP

The digested pSB1C3 was re-purified in order to get rid of any contaminant DNA that arose during the digestion.

  • Restriction digests of pveg and Spec-T

pveg (promoter and RBS) and Spec-T (Spectinomycin with a terminator) were digested in preparation for 3A assembly.

  • Ligation of 5' ins (ES) and dif (XP) with pSB1C3 (EP)

The digested 5' ins and dif (the front inserts) were ligated overnight with pSB1C3 (the vector). A bench ligation and an overnight ligation were set up.

  • Transformation of E.Coli with bench ligate

E.Coli was transformed via the chemical method using the bench ligate.

  • Gel extraction and purification of pveg and Spec-T

Since these are both inserts they were gel extracted and purified. PCR purification is not carried out for inserts since they are small and would therefore be lost during the process.

Wednesday, August 18
  • Replica plating and colony PCR of 5' ins and dif in pSB1C3 (bench ligation)
  • Ligation of pveg (ES) and SpecR-T (XP) with pSB1C3 (EP)

pveg and SpecR-T (the front inserts) were ligated overnight with pSB1C3 (the vector).

Thursday, August 19
  • Transformation of E.Coli with the overnights ligates
  1. 5' ins and dif in pSB1C3
  2. pveg and SpecR-T in pSB1C3
Friday, August 20
  • Replica plating and colony PCR of both transformations from yesterday.
  • Annealing the forward and reverse strands of the dif P ES oligo

The forward and reverse strands of the 3' dif Pme1 sites with XbaI and PstI restriction sites on either side have been synthesized separately. The synthesized fragments arrive in solid powder form. These were immediately diluted in ddH2O to obtain a stock concentration of 1 ng/ul. They were then allowed to anneal together by heating them to 95 degs for denaturation and allowing them to cool down and anneal overnight.

Week 8

Week 8 Monday Tuesday Wednesday Thursday Friday Saturday
Morning
  • Set up overnight ligations for standard assembly (BBA) and 3A cloning (3A) of dif P
  • Restriction digestion of 3' ins in the pSB1AK3 vector ( Using Eco and Xba) for BBA
  • PCR purification of 3' ins in AK3 ( Using Eco and Xba) for BBA
  • Set up 5 ml culture of pveg, SpecR-T in pSB1C3 from colony 1 of replica plate and kept in shaking incubator at 37 degrees
  • Restriction digestion of 3' ins in the AK3 vector ( Using Xba and Pst) for 3A
    • Overnight ligation of dif P and 3' ins with pSB1C3
    • Transformations using the overnight ligations showed a lot of background. Therefore we set up ligations for K02 and K09 using 3A cloning. The results will show if this method is preferable due to less background.
  • Replica plating of transformed colonies having dif P with 3' ins in AK3 -45 sigle colonies were plated
  • The first 15 of the above colonies were used for colony PCR reactions using dif PES Fwd and pSB Rev
  • Gel analysis of colony PCR from yesterday (first 15 replica plated colonies
  • Set up further colony PCR reactions for the same 15 colonies using pSB Fwd and pSB Rev primers
  • Miniprep of 4 overnight cultures - diff P with 3' ins in AK3; colonies 4,5,7 & 9
Afternoon
  • Gel analysis of PCR purified 3' ins in AK3 and gel purified 3' diff P to work out ratios for the liagation
  • Dephosphorylation of digested AK3 vector with 3' ins
  • Overnight ligation of dif P with 3' ins in AK3 (insert and vector) and AK3 containing 3' ins vector only
  • Set up 100 ml culture of pveg and Spec-T in pSB1C3 by transferring 5 ml culture set up in the morning and kept in shaking incubator at 37 degrees
  • Transformation of E.Coli with ligates from yesterday in AmpR;dif P with 3' ins in AK3 (insert and vector) and AK3 containing 3' ins vector only
  • Gel extraction of 3' ins (now our insert) described this morning for 3A
  • Gel analysis of gel extracted dif P and 3' ins (inserts) and pSB1C3 (vector) to work out ratios for the ligation
  • Midiprep of pveg, SpecR-T in pSB1C3 from colony
  • Gel analysis of gel extracted dif P and 3' ins (inserts) and pSB1C3 (vector) to work out ratios for the ligation
  • pveg, SpecR-T in pSB1C3 midiprep sent for sequencing
  • Set up overnight 5 ml cultures containing dif P with 3' ins in AK3 for miniprep tomorrow - 4 cultures were set up by looking at the gel this morning; 2 positive looking (4 & 7), 1 negative (5) and one containing nothing (9
  • Diagnostic digests of minipreps containing dif P with 3' ins in AK3 - Two digests : One with Spe & Pst and other with Xba & Spe

Week 9

Week 9 Monday Tuesday Wednesday Thursday Friday
Morning
  • Gel analysis of Mini preps and diagnostic digests from Saturday - Colony 4 looked best
  • Midiprep of Colony 4 - concentration 110 ng/ul
    • Gel purification of insert (35 ul)
    • Gel analysis of vector and insert to work out ratios for ligations
  • Transformation with overnight ligations
  • The transformations were highly successful!! The Vector only plate showed no colonies and the Insert & Vector plate showed many individual colonies
  • 10 individual colonies were replica plated and used for colony PCR
Afternoon
  • Set up 200 ml overnight culture of colony 4 (containing Dif P and K09) for midiprep tomorrow
  • Digests set up for Vector (SpecR) with Spe & Pst and Insert (dif P & K09)with Xba & Ps
  • PCR purification of vetor (35 ul)
  • Gel extraction of insert after gel analysis (35 ul)
  • Dephosphorylation of vector
  • Set up two overnight ligations; Vector & Insert and Vector only
  • Both ligations (Insert & Vector and Vector only) were plated in CmR and incubated overnight
  • Gel analysis of colony PCR products

Week 10

Week 10 Monday Tuesday Wednesday Thursday Friday
Morning
  • Colony PCR of 10 transformed C-Spec colonies
  • Set up 5ml culture of N-Spec in KanR
  • Miniprep of C-Spec colonies 2, 5 & 9 in SpecR and CmR
  • Digest of minipreps; double digest (Eco and Spe) and triple digest (PmeI, Eco and Spe)
  • Gel analysis of midiprepped undigested and digested C-Spec
    • Miniprep of C-Spec colonies 2, 5 & 9 in SpecR only and CmR only
    • Digest of minipreps; double digest (Eco and Spe) and triple digest (PmeI, Eco and Spe)
    • Gel analysis of midiprepped undigested and digested C-Spec
  • Start midiprep of C-Spec from colony 2 (isopropanol added elute was refrigerated for 4 hrs)
  • Measure concentration of midiprepped N-Spec (732 ng/ul)
  • Dilution of N-Spec and C-Spec (4x)
  • Double digest of N-Spec and C-Spec (Eco and KpnI)
  • Gel analysis of diluted undigested and double digested Specs
Afternoon
  • Gel analysis of colony PCR
  • Set up 5 ml cultures of C-Spec colonies 2,5 & 9 in SpecR & CmR
  • Transfer 5 ml culture to 100 ml culture in KanR (incubated overnight)
  • Start midiprep of N-Spec (isopropanol added elute refrigerated overnight)
  • Set up 5 ml cultures for C-Spec from colonies 2,5 & 9 (in SpecR only and CmR only)
  • Continue midiprep of N-Spec (by ethanol preciptation)
  • Set up a 500 ml overnight culture (with SpecR) of C-Spec from colony 2 for a midiprep tomorrow
  • Continue midiprep of C-Spec (by ethanol precipitation)
  • Digest of midiprepped N-Spec and C-Spec (Eco and Spe)
  • Gel analysis of undigested and digested (Eco and KpnI) Specs
  • Single digest of diluted C-Spec (Eco only)
  • Gel analysis of diluted undigested, single digested (Eco only) and double digested (Eco and KpnI)


Week 11

Week 11 Monday Tuesday Wednesday Thursday Friday
Morning
  • Set up digests of N-Spec and C-Spec (Eco and KpnI)- (CD 1)
  • Gel analysis and extraction of both N and C Specs
  • Gel purification of N and C Specs from CD 2
  • Run both Specs from each of the ligations on a gel
    • Backbone PCR of pSB1C3 vector using PFU ultra buffer
    • PCR purification of pSB1C3
  • Backbone PCR of pSB1C3 using Barns buffer
  • Replica plating in SpecR of transformed colonies from the insert and vector ligation and setting up 5 ml cultures in SpecR for transformed colonies
  • Gel purification of pSB1C3
  • Miniprep of 5 ml overnight cultures of colonies 1, 2 and 3
Afternoon
  • Set up digests of N-Spec and C-Spec again, however, with a higher dilution of N-Spec - (CD 2)
  • Gel analysis and extraction of N and C Specs
  • Gel purification of CD 1
  • Dephosphorylate vector (C-Spec) from CD 2
  • Set up overnight ligations of Vector only (C-Spec) and Vector and Insert (N-Spec and C-Spec) for transformation
  • Transformation of E.Coli with the two Spec final ligates - C-Spec (vector only) and N and C Specs (vector and insert)
  • Gel analysis of purified pSB1C3
  • Gel analysis and extraction of pSB1C3
  • Test digest of final spec minipreps with Eco and Kpn1 and Eco and Spe
  • Cloning digest of pSB1C3 with Eco and Pst
  • Gel analysis of undigested and digested minipreps and digested pSB1C3

Week 12

Week 12 Monday Tuesday Wednesday Thursday Friday Saturday
Morning
  • Set up 5 ml culture for miniprep of C-Spec of colnies 1 and 2 from synthesis in AmpR
  • Set up sequencing mixes for 3' dif P Ins in AK3, C-Spec minis from cultures 5 & 9 and final spec mini
  • Start midiprep of C-Spec
  • Miniprep of C-Spec cultures from yesterday
  • Start midiprep of C-Spec colonies again
    • Cloning digests of C-Spec with Kpn1 and Pst and N-Spec with Eco and Kpn1
    • Gel purification of the inserts
    Afternoon
    • Miniprep cultures of C-Spec have not grown well enough therefore they will be left overnight
    • Set up 100 ml culture for midiprepping tomorrow
    • Test digest C-Spec mini with ECo and KpnI
    • Run test digest on gel, both minis from colonies 1 & 2 look good, therefore proceed to midiprep
    • Continue with midiprep but no pellet after 45 minute spin
    • Set up 100 ml cultures of colonies 1 & 2 for midiprep tomorrow
    • Continue Midiprep of C-Spec, pellets obtained
    • Transformation of E.Coli with the two Spec final ligates - C3 (vector only) and N and C Specs in C3 (vector and insert)
    • Ligation gel with inserts; N-Spec (E & Kpn1) and C-Spec (Kpn1 & P) and Vector; pSB1C3 (E & P)
    • Dephosphorylation of Vector and overnight ligation of vector alone and vector with inserts
    • Transformation of E.Coli with the two Spec final ligates - C3 (vector only) and N and C Specs in C3 (vector and insert)
    • Transformations failed, re-try next week!

    Week 13

    Week 13 Monday Tuesday Wednesday Thursday Friday Saturday
    Morning
    • Set up 5 ml culture for miniprep of C-Spec of colnies 1 and 2 from synthesis in AmpR
    • Set up sequencing mixes for 3' dif P Ins in AK3, C-Spec minis from cultures 5 & 9 and final spec mini
    • Start midiprep of C-Spec
    • Miniprep of C-Spec cultures from yesterday
  • Start midiprep of C-Spec colonies again
    • Cloning digests of C-Spec with Kpn1 and Pst and N-Spec with Eco and Kpn1
    • Gel purification of the inserts
    Afternoon
    • Miniprep cultures of C-Spec have not grown well enough therefore they will be left overnight
    • Set up 100 ml culture for midiprepping tomorrow
    • Test digest C-Spec mini with ECo and KpnI
    • Run test digest on gel, both minis from colonies 1 & 2 look good, therefore proceed to midiprep
    • Continue with midiprep but no pellet after 45 minute spin
    • Set up 100 ml cultures of colonies 1 & 2 for midiprep tomorrow
    • Continue Midiprep of C-Spec, pellets obtained
    • Transformation of E.Coli with the two Spec final ligates - C3 (vector only) and N and C Specs in C3 (vector and insert)
    • Ligation gel with inserts; N-Spec (E & Kpn1) and C-Spec (Kpn1 & P) and Vector; pSB1C3 (E & P)
    • Dephosphorylation of Vector and overnight ligation of vector alone and vector with inserts
    • Transformation of E.Coli with the two Spec final ligates - C3 (vector only) and N and C Specs in C3 (vector and insert)
    • Transformations failed, re-try next week!

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