Franquicias en Puebla

2.10.1. Single stranded plasmid DNA preparation (ssDNA)

Colonies of interest for sequencing of plasmid or phage DNA were picked using a sterile cocktail stick into a glass tube containing 1.5ml o f 2 x TY. If plasmid DNA was to be rescued, ampicillin at a concentration of lOOpg/ml was added. No antibiotic was required for the M13 cultures. These cultures were then incubated at 37°C in a shaking incubator overnight.

The following morning new glass tubes containing 1.5ml of 2 x TY (plus antibiotic if required) were prepared. A 1:100 dilution from the overnight culture was then set up the following day. The tubes were then returned to the 37°C incubator. The M13 cultures were grown for 5.5 hours. The plasmid cultures were grown for 2 hours and then 3pl of Helper Phage (VCS-M13, Stratagene catalog no. 200251-81) was added to each tube to rescue the single stranded copy of the DNA. The cultures were then incubated for another 5.5 hours at 37°C with vigorous shaking.

The cultures were then transferred to 1.5ml Eppendorf tubes and spun for 5 minutes at maximum speed in a MSE Microcentaur microfuge. The supernatant was transferred to a new 1.5ml tube containing 200|xl of 20% PEG 2.5M NaCl. The tubes were then left at room temperature for a minimum of 10 minutes before spinning again at maximum speed for 5 minutes. The pellet was carefully dissolved in lOOpl of TE and 200pl of PCIA was added. The mixture was vortexed for 5 seconds before spinning at 13000 rpm for 5 minutes to collect the aqueous phase. The aqueous phase was transferred to a fresh tube and the previous step was repeated. lOOpl of CIA was then added to the aqueous phase to remove residual phenol. The two layers were mixed by vortexing and the tubes were then spun again. The top layer was transferred to a new tube and 10% Na- Acetate and 250p,l 100% ethanol were added to precipitate the single stranded DNA (ssDNA). The DNA was incubated on ice for at least 20 minutes before being recovered by centrifugation at 13000 rpm for 5 minutes. The pellet was washed with 70% ethanol and then air dried. Each pellet was then resuspended in 30pl TE and stored at -20°C until required.

2.10.2. Manual DNA sequencing

The dideoxy chain termination method for manual sequencing was originally described by Sanger era/. (Sanger et a/., 1977). All sequencing reactions were performed in U bottom microtitre plates. 2pl of ssDNA from each clone were transferred into 4 vertical wells. 2|xl of the following master mix were then added to each well; Ifxl reaction buffer, Ip l M13 -40 primer and 6pl water. The plates were then spun briefly in a MSE Mistral 2000 centrifuge to collect the reaction mix at the bottom of the well, covered in Saran wrap and incubated at 55°C for 30 minutes to allow the primer to anneal to the template. During this time a mix was made up for each clone as follows;

0.4pl 7.5pM dNTP 0.4pl lOOmMDTT 6.5pl water

35S-dATP 0.5 pi

0.25pl sequenase enzyme (Amersham Life Sciences 13U/ml)

After incubation at 55®C, 2pl of the above solution were transferred to each well. The plates were spun again and incubated at room temperature for 10 minutes. 2pl of one of four stop nucleotide mixes (Amersham Life Sciences 250ul) i.e. T, C, G or A were added to each of the four wells. The plates were briefly spun as before and incubated at 37®C for 6 minutes. The reactions were terminated by adding 4pl of formamide stop solution to each well. The reactions were incubated at 80°C for 20 minutes to denature the DNA, prior to loading the sequencing gel.

M l 3 -40 primer sequence

5'- GTTTTCCCAGTCACGAC -3'

2.10.3. Acrylamide gel electrophoresis for sequencing

Sequencing electrophoresis was performed using 40 x 50 x 0.4cm gradient gels. Before pouring the gel, the plates were siliconised with Repelcote. The plates were separated by 0.4mm spacers and fastened together with tape and bulldog clips. 30pl of 10% ammonium persulphate solution (APS) and 13pl of Temed (Amresco) was added to 15ml of 2.5 x TBE acrylamide gel mix and the reaction

was poured into the plates using a 50ml syringe. This was followed by the second mix of 50ml 0.5 x TBE acrylamide gel mix, lOOjil APS and 65[xl Temed. A comb to form the wells was placed in position and the gel was allowed to set for 30 minutes. The tape and bulldog clips were then removed and the gel was then placed in a Gibco BRL S2 gel tank, 1 litre of 0.5 x TBE was used as buffer and 4pl of each sample reaction loaded into each well. The gel was run at 30 volts for 3.5-4 hours. The plates were then disassembled and the back plate was removed. The acrylamide gel was transferred to 3MM Whatman paper and covered with Saran wrap before being dried under vacuum at 80°C on a gel dryer for 2 hours. The dried gel was then exposed overnight at room temperature to Fuji Medical autoradiography film. Sequences were analysed using DNAStar computer software.