IGEM:Imperial/2010/AmyE Vector

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Starting from the top, we are assembling the first two fragments (K14070 and K14064) and K147002 with our oligos to add in a Dif site. Two dif sites on either side of resistance cassettes can be used to later excise antibiotic resistance from our final constructs.

A : K143070 and K11064 assembly step
A : K143070 and K11064 assembly step
  • K14070 and K14064 fragments
- DNA was taken out of the reigstry
- Cut with restriction enzymes
- Run on a gel to confirm correct cutting
  and estimate relative ratios of DNA for ligation
- Ligated overnight
- Transformed into E.Coli to Amplify the DNA
- Colony PCR has been used to confirm the correct insert size.
  • Next Steps:
- Extract the DNA with a miniprep
- Proceed to the next step - Reverse PCR


B : K14002 and oligo assembly
B : K14002 and oligo assembly






  • K14002 and oligos
- Ligated two single stranded oligos together to produce Dif and insertion site
- Standard biobrick assembly of oligos to K14002
- Ligation and transformation into E.coli competent cells (strain)
- Screen plated colonies for correct insertion
  • Next Steps
- Purify the correct insert out of E.coli
- Next step assembly - LacI test vector and Final assembly vector









Image:LacI testing construct
B2 : LacI Testing Vector
  • LacI Testing vector
- Currently waiting for Part B step 2 Midi-prep results to start this step



Contents

Schedule & Lab notes

Week 7

Week 6 Monday Tuesday Wednesday Thursday Friday
MORNING Starting assembly of AmyE vector
  • Restriction digestion of BB k14070 for the AmyE vector (using Eco and Spe)
  • Restriction digestion of BB k14064 for the AmyE vector (using Eco and Xba)
  • Restriction digestion of 5' integration site (k08) for the PyrD vector (using Eco and Spe)
  • PCR amplification of vector backbone PSB1C3 for the PyrD vector
  • Gel analysis and extraction of 5' int site for PyrD vector (repetition of step due to absence of DNA during gel analysis yesterday)
  • Gel purification of k70
  • Gel purification of k08
  • Re-analysis of k70, k64 (for AmyE) on gel to work out ratios for ligation set up
  • Re-analysis of k08, Pme oligos and pSB1C3 (for PyrD) on gel to work out ratios for ligation set up
  • Restriction digestion of pveg promoter (k53) (using Eco and Spe) and spec cassette (k65) (using Xba and Pst) for PyrD vector
  • Restriction digestion of pSB1C3 for PyrD (using Eco and Pst)
  • Check for transformed colonies (colonies that have taken up the vector with the 5'diff) and prepare for colony PCR
  • Gel purification of k53 and k65 for AmyE in preparation for ligations this afternoon
  • Gel extraction and re-analysis on gel of k53, k65 and psB1C3 for Spec casette in preparation for ligations this afternoon
  • Transformation of overnight ligations of:

k64 and k70, k70 only, Spec and 5' PyrD diff

  • Replica plating and colony PCR of:

Spec and 5' PyrD diff

  • Plate wash of:

k64 and k70. k70 only was discarded since this was purely for a baground check

AFTERNOON
  • PCR purification of PSB1C3 vector
  • PCR purification of BB k14064 digestion products
  • Gel analysis of digestion products of BB k14070
  • Gel extraction of digestion products of BB k14070
  • Restriction digestion of k64 and subsequent PCR purification (repetition of step due to absence of DNA during gel analysis)
  • Gel analysis of k70, k64 (for AmyE) to work out ligation ratios
  • Gel analysis and extraction of k53 and k65
  • Bench (1 hour) and overnight ligation of 5'diff with the pSB1C3 vector
  • Transformation of E.Coli with the bench ligated vector
  • Dephosphorylation of k64 and set up of overnight ligations for k64 and k70 (vector and insert) and k64 (vector)only (for negative control; check of background)
  • Set up overnight ligations of SpecR
  • Gel analysis of colony PCR products of Spec and 5' PyrD diff
  • Annealing of diff P oligos (used for both PyrD and AmyE vectors)

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' integration site (K02) in the A2 vector ( Using Eco and Xba) for BBA

  • Restriction digestion of 3' integration site (K09) in the AK3 vector ( Using Eco and Xba) for BBA

  • PCR purification of 3' integration site (K02) in the A2 vector ( Using Eco and Xba) for BBA

  • PCR purification of 3' integration site (K09) in the AK3 vector ( Using Eco and Xba) for BBA

  • Set up 5 ml culture of spec from colony 1 of replica plate in shaking incubator @ 37 degrees

  • Restriction digestion of 3' integration site (K02) in the A2 vector ( Using Xba and Pst) for 3A

  • Restriction digestion of 3' integration site (K09) in the AK3 vector ( Using Xba and Pst) for 3A

  • 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.
  • Midiprep of CmR vector and test digest using Eco and Spe
  • Backbone PCR of PSB1C3 vector (1st attempt) for common use


  • Replica plating of transformed colonies for k09 from the plate with the insert (diff P) - 45 sigle colonies were plated
  • The first 15 of the above colonies were colony PCRed 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
  • Gel analysis of pSB1C3 - after backbone PCR and after digestion (looked contaminated!)
  • Test digests of K54 K70 Midi prep using i) EcoRI, ii) SpeI and iii) EcoRI + SpeI
  • Screen next 20 colonies by colony PCR, use higher temperatures to avoid previous non specific annealing
  • Miniprep of 4 overnight cultures (dif P and 3' Insert for PyrD) - colonies 4,5,7 & 9
  • Run Colony PCR results on a Gel - pick promissing candidates for mini prep.
AFTERNOON
  • Gel analysis of PCR purified K02 and K09 with the insert (diff P) in between to work out ratios for the liagation
  • Dephosphorylation of digested A2 vector with 3' integration site (K02)

  • Dephosphorylation of digested AK3 vector with 3' integration site (K09)

  • Set up overnight ligation of A2 vector with 3' integration site (K02) with diff P (insert) and A2 vector only

  • Set up overnight ligation of AK3 vector with 3' integration site (K09) with diff P (insert) and AK3 vector only
  • Colony PCR and gel analysis of plated culture (from plate wash on Friday) with K64 and K70
  • Overnight 100 ml culture of spec @ 14 degrees
  • Electroporation of the 4 overnight ligations described on Thursday afternoon
  • Gel extraction of the digestion products ( 3' integration sites - now our inserts) described this morning for 3A
  • Gel analysis of gel extracted K02 nad K09 (inserts) with diff P (also an insert) and pSB1C3 (vector) in between
  • Set up overnight 100 ml culture of CmR vector (K64 and K70) for midiprep tomorrow
  • Set up overnight culture plates (AmpR) for the 4 electroporated cultures (colonies that survive will contain transformed cells
  • PCR purification of PSB1C3 PCR amplified vector
  • Check concentration of midi prepped CmR
  • Run a gel to visualise the results - Gel contained pSB1C3 (PCR purified) , CmR (Midiprepped) and CmR digested (Midiprepped)
  • Backbone PCR of pSB1C3 (2nd attempt), PCR purified and then digested with Eco and Pst
  • Midipreps sent for sequencing ( Spec and CmR)

Backbone PCR of pSB1C3 using PFU (3rd attempt)

  • Set up overnight 5 ml cultures for miniprepping 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 - Two digests : One with Spe & Pst and other with Xba & Spe

Week 9

Week 8 Monday Tuesday Wednesday Thursday Friday
MORNING
  • Gel analysis of Mini preps and diagnostic digests from Saturday - Colony 4 looked best
  • Test Digest of Mini Prep K02+dif EcoRI and SpeI
  • Midiprep of Colony 4 - concentration 110 ng/ul
  • Restriction digest of K02 from registrty for 3A assembly.
  • Gel purification of insert (35 ul)
  • Gel analysis of vector and insert to work out ratios for ligations
  • Transformation with overnight ligations
  • PCR amplified the PSB1C3 vector for 3A assembly
  • Gel Purified K02 and PBB1C3 digests
  • 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
  • Dephosphorylate PSB1C3 using alkaline phosphotase
AFTERNOON
  • Set up 200 ml overnight culture of colony 4 (containing Dif P and K09) for midiprep tomorrow
  • Second Test digest SpeI PME - no positive results. Decided to repeat the step using 3A assembly method to reduce background from vector.
  • Digests set up for Vector (SpecR) with Spe & Pst and Insert (dif P & K09)with Xba & Pst
  • PCR purification of vetor (35 ul)
  • Gel extraction of insert after gel analysis (35 ul)
  • Midi prep of sample 8 K54 + K70
  • 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
  • set up ligation reaction K02+dif using 3A method for Transformation on Monday.

Week 10

Week 8 Monday Tuesday Wednesday Thursday Friday
MORNING
  • Gel analysis of Mini preps and diagnostic digests from Saturday - Colony 4 looked best
  • Test Digest of Mini Prep K02+dif EcoRI and SpeI
  • Midiprep of Colony 4 - concentration 110 ng/ul
  • Restriction digest of K02 from registrty for 3A assembly.
  • Gel purification of insert (35 ul)
  • Gel analysis of vector and insert to work out ratios for ligations
  • Transformation with overnight ligations
  • PCR amplified the PSB1C3 vector for 3A assembly
  • Gel Purified K02 and PBB1C3 digests
  • 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
  • Dephosphorylate PSB1C3 using alkaline phosphotase
AFTERNOON
  • Set up 200 ml overnight culture of colony 4 (containing Dif P and K09) for midiprep tomorrow
  • Second Test digest SpeI PME - no positive results. Decided to repeat the step using 3A assembly method to reduce background from vector.
  • Digests set up for Vector (SpecR) with Spe & Pst and Insert (dif P & K09)with Xba & Pst
  • PCR purification of vetor (35 ul)
  • Gel extraction of insert after gel analysis (35 ul)
  • Midi prep of sample 8 K54 + K70
  • 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
  • set up ligation reaction K02+dif using 3A method for Transformation on Monday.

Week 11

Week 8 Monday Tuesday Wednesday Thursday Friday
MORNING
  • Gel analysis of Mini preps and diagnostic digests from Saturday - Colony 4 looked best
  • Test Digest of Mini Prep K02+dif EcoRI and SpeI
  • Midiprep of Colony 4 - concentration 110 ng/ul
  • Restriction digest of K02 from registrty for 3A assembly.
  • Gel purification of insert (35 ul)
  • Gel analysis of vector and insert to work out ratios for ligations
  • Transformation with overnight ligations
  • PCR amplified the PSB1C3 vector for 3A assembly
  • Gel Purified K02 and PBB1C3 digests
  • 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
  • Dephosphorylate PSB1C3 using alkaline phosphotase
AFTERNOON
  • Set up 200 ml overnight culture of colony 4 (containing Dif P and K09) for midiprep tomorrow
  • Second Test digest SpeI PME - no positive results. Decided to repeat the step using 3A assembly method to reduce background from vector.
  • Digests set up for Vector (SpecR) with Spe & Pst and Insert (dif P & K09)with Xba & Pst
  • PCR purification of vetor (35 ul)
  • Gel extraction of insert after gel analysis (35 ul)
  • Midi prep of sample 8 K54 + K70
  • 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
  • set up ligation reaction K02+dif using 3A method for Transformation on Monday.

Week 12

Week 8 Monday Tuesday Wednesday Thursday Friday
MORNING
  • Gel analysis of Mini preps and diagnostic digests from Saturday - Colony 4 looked best
  • Test Digest of Mini Prep K02+dif EcoRI and SpeI
  • Midiprep of Colony 4 - concentration 110 ng/ul
  • Restriction digest of K02 from registrty for 3A assembly.
  • Gel purification of insert (35 ul)
  • Gel analysis of vector and insert to work out ratios for ligations
  • Transformation with overnight ligations
  • PCR amplified the PSB1C3 vector for 3A assembly
  • Gel Purified K02 and PBB1C3 digests
  • 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
  • Dephosphorylate PSB1C3 using alkaline phosphotase
AFTERNOON
  • Set up 200 ml overnight culture of colony 4 (containing Dif P and K09) for midiprep tomorrow
  • Second Test digest SpeI PME - no positive results. Decided to repeat the step using 3A assembly method to reduce background from vector.
  • Digests set up for Vector (SpecR) with Spe & Pst and Insert (dif P & K09)with Xba & Pst
  • PCR purification of vetor (35 ul)
  • Gel extraction of insert after gel analysis (35 ul)
  • Midi prep of sample 8 K54 + K70
  • 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
  • set up ligation reaction K02+dif using 3A method for Transformation on Monday.

Week 13

Week 8 Monday Tuesday Wednesday Thursday Friday
MORNING
  • Gel analysis of Mini preps and diagnostic digests from Saturday - Colony 4 looked best
  • Test Digest of Mini Prep K02+dif EcoRI and SpeI
  • Midiprep of Colony 4 - concentration 110 ng/ul
  • Restriction digest of K02 from registrty for 3A assembly.
  • Gel purification of insert (35 ul)
  • Gel analysis of vector and insert to work out ratios for ligations
  • Transformation with overnight ligations
  • PCR amplified the PSB1C3 vector for 3A assembly
  • Gel Purified K02 and PBB1C3 digests
  • 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
  • Dephosphorylate PSB1C3 using alkaline phosphotase
AFTERNOON
  • Set up 200 ml overnight culture of colony 4 (containing Dif P and K09) for midiprep tomorrow
  • Second Test digest SpeI PME - no positive results. Decided to repeat the step using 3A assembly method to reduce background from vector.
  • Digests set up for Vector (SpecR) with Spe & Pst and Insert (dif P & K09)with Xba & Pst
  • PCR purification of vetor (35 ul)
  • Gel extraction of insert after gel analysis (35 ul)
  • Midi prep of sample 8 K54 + K70
  • 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
  • set up ligation reaction K02+dif using 3A method for Transformation on Monday.
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