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
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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
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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)
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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
5' ins and dif in pSB1C3
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
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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
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Week 9
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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
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Week 10
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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)
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Week 11
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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
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Week 12
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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!
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Week 13
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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)
