3.2.1.TISSUE COLLECTION
Wild type mice were killed by Schedule One procedure and relevant tissues were quickly removed and frozen down on dry ice. Hypothalami were separated from the rest of the brain tissue by dissection for analysis of this brain region. It is particularly important the tissues are frozen down immediately to maintain RNA integrity. Tissues were stored at -80°C and used for RNA extraction.
3.2.2.RNAEXTRACTION
RNA was extracted from 40 mg hypothalamic tissue samples using the Stratagene RNA extraction kit following the protocol for tissue extraction. This included homogenization of tissue samples with 600 µL lysis buffer containing 4.2 µL β-Mercaptoethanol. Homogenate was added, 700 μL at a time, to a pre- filter spin-cup in a 2 mL receptacle tube and centrifuged for 5 minutes. The filtrate was added to an equal volume of 70% EtOH and mixed thoroughly. From the filtrate/EtOH mixture, 700 µL was transferred to an RNA-binding spin-cup and centrifuged for 1 minute, the filtrate was discarded and the rest of the mixture added to the spin-cup and centrifuged for 1 minute.
If RT-PCR was to be performed on the RNA extracts, 600 µL 1 x low salt buffer was added to the spin-cup and centrifuged for 1 minute. The cup was retained and replaced in the receptacle tube (the filtrate was discarded), 50 µL RNAse-free DNAse digestion buffer and 5 µL reconstituted RNAse-free DNAse I was pipetted onto the fibre matrix and incubated for 15 minutes at 37°C. 600 µL 1 x high salt wash buffer was added to the spin-cup and centrifuged for 1 minute. The filtrate was discarded and the spin-cup replaced in the receptacle
tube. 600 µL 1 x low salt wash buffer was added and the tube centrifuged for 1 minute. Again the spin-cup was retained and the filtrate discarded. 300 µL 1 x low salt buffer was added and the tube centrifuged for 2 minutes. The spin-cup was transferred to a fresh 1.5 mL tube and 30 µL elution buffer was added to the fibre matrix of the spin-cup, incubated for 2 minutes at room temperature and centrifuged for 1 minute. The elution step was repeated, giving a final volume of 60 μL eluate. These RNA extracts were stored at -80°C.
3.2.3.REVERSE TRANSCRIPTION-PCR(RT-PCR)
For each sample 1 μg total RNA was required. Hexamer stock (0.6 μL of a 500 ng/μL) was added and the volume made up to 13.7 μL with RNAse-free water. This was incubated at 65°C for 5 minutes then cooled slowly to room temperature (20°C). To each reaction 2 μL 10x AffinityScript™ RT buffer; 2 μL 100 mM DTT; 0.8 μL 10 mM dNTP mix; 0.5 μL RNAse inhibitor (20U); and 1 μL AffinityScript™ Multiple Temperature Reverse Transcriptase (Stratagene) were added. The reaction was mixed gently, incubated at 25°C for 10 minutes; 42°C for 1 hour; 70°C for 15 minutes and cooled to 37°C. 1 μL RNAseH was added and the reactions were incubated at 37°C for 20 minutes. The cDNA obtained was stored at -20°C.
PCR reactions consisted of 1 µL cDNA, 22 µL ddH2O, 3 µL PCR buffer, 3 µL
dNTPs, 0.5 µL forward primer, 0.5 µL reverse primer and 0.25 µL Taq polymerase. PCR reactions were run on the following thermocycler programme: heated lid at 111°C, 15 minute denaturation at 95°C, this was followed by 30 cycles of 45 seconds at 95°C (denaturation), 45 seconds at 56°C (annealing) and 1 minute at 72°C (extension). PCR products were stored at 4°C until required.
Chapter 3. Analysis of Gnasxl Exon A20 Splicing
3.2.4.LIGATION AND CLONING INTO COMPETENT E. COLI
Equimolar amounts of supercoiled, uncut plasmid (~50 ng) and PCR products were used in a total volume of 25 μL along with 12.5 μL H2O; 1 x
Ligation buffer; 0.2 mM dNTP, 1U SmaI restriction enzyme; 1 U T4 DNA Polymerase; and 3U T4 DNA ligase. This was left at room temperature overnight. One third of the reaction was transformed into competent E. coli (Strain: DH5 ). Bacteria were grown on LB + Ampicillin (Amp) plates at 37 C overnight.
3.2.5. CLONING OF PCR PRODUCTS USING THE TOPO TA CLONING® KIT
(INVITROGEN)
A TOPO TA Cloning kit was used to clone PCR products into competent bacterial cells. Two LB + Amp plates per cloning reaction to be performed were pre-warmed to 37 °C. 4 μL amplified PCR product to be cloned into bacteria was added to 1 μL salt solution (1.2 M NaCl; 0.06 M MgCl2) and 1 μL 10 ng/μL pCR
2.1-TOPO vector and incubated at room temperature for 5 minutes. To transform the vector into bacterial cells, 2 μL of the TOPO TA cloning ® reaction mixture was added to one vial of thawed, one-shot TOP10 cells and incubated on ice for 30 minutes. The TOP10 cells were then heat-shocked for 30 seconds at 42°C and immediately placed on ice. 250 μL SOC medium was added to the TOP10 cells in a mini-culture tube. This was left at 37 C for 1 hour with constant agitation. These were then plated out in 50 μL and 200 μL aliquots onto the pre-warmed LB + Amp plates and incubated at 37 C overnight.
3.2.6.PREPARATION OF BACTERIAL MINI-CULTURES
Individual colonies were selected from cloning experiment LB + Amp plates and grown overnight in individual mini-culture tubes containing 2 mL LB
+ Amp broth at 37 C with constant agitation. After overnight incubation these were stored at 4 C and could be used to re-grow colonies of interest if necessary.
3.2.7. TENS MINI-PREP FOR EXTRACTION OF DNA FROM BACTERIAL MINI- CULTURES
Overnight bacterial mini-cultures were tipped into 1.5 mL tubes and spun in the centrifuge for 30 seconds at full speed. The supernatant was discarded, leaving a small amount (~50 μL) in the centrifuge tube for resuspension of the pellet. 300 μL of TENS buffer was added to lyse cells. Samples were placed on ice for 5 minutes. 150 μL of 3 M sodium acetate, pH 5.0, was added and mixed immediately by inverting. The samples were returned to ice for 5 minutes then centrifuged at maximum speed for 5 minutes. The supernatant was transferred to a separate tube containing 900 μL 100% EtOH, mixed and placed at -20°C for 30 minutes. Samples were spun for 5 minutes and the supernatant was removed. Pellets were washed twice with 700 μL 70% EtOH with a 1 minute spin. The EtOH was removed and the pellets allowed to dry for 10 minutes at 37°C then resuspended in 30 μL 1 x TE + RNAseA. These were stored at 4°C.
3.2.8.DETERMINATION OF COLONIES CONTAINING PCR PRODUCT INSERTIONS BY
ETBR AGAROSE GEL ELECTROPHORESIS
Mini-prep DNA was digested using restriction enzymes to cut out the PCR product insert; 5 μL digested mini-prep DNA was run on 2% agarose gels containing EtBr. Colonies that were deemed to contain fragments of interesting size after visualisation on the gel doc were sent for sequencing by DBS
Chapter 3. Analysis of Gnasxl Exon A20 Splicing
the full-length sequence to determine exon splicing of the fragment using NCBI BLAST.