IGEM:Imperial/2010/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

• 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)

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.

• 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

• 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

• 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.

• 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

• 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.

• 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

• 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.

• 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

• 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.

• 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

• 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.