IGEM:Imperial/2010/PyrD Vector

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

Friday, August 13
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.
 * Restriction digest of 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.
 * PCR purification of the cut dif XP


 * Gel Analysis of dif XP

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

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 amplification 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.
 * PCR purification of pSB1C3

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

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

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

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

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

E.Coli was transformed via the chemical method using the bench ligate.
 * Transformation of E.Coli with bench ligate

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.
 * Gel extraction and purification of pveg and Spec-T

Wednesday, August 18

 * Replica plating and colony PCR of 5' ins and dif in pSB1C3 (bench ligation)

pveg and SpecR-T (the front inserts) were ligated overnight with pSB1C3 (the vector).
 * Ligation of pveg (ES) and SpecR-T (XP) with pSB1C3 (EP)

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.

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.
 * Annealing the forward and reverse strands of the dif P ES oligo