1. A vial of frozen cells stored in liquid N2 was thawed quickly (under a running tap). 2. The cells were transferred to a 15ml falcon tube and centrifuged at 1 lOOrpm for 5 minutes. 3. The supernatant was removed and the cells were resuspended in 2ml of Modified eagle medium, supplemented with 10% fetal bovine serum, 2% L-Glutamine and 2% Penicillin-Streptomycin. 4. Cells were again centrifuged and resuspended as before (steps 2 and 3).
5. Cells were added to 7ml of the above media, in a 75cm^ flask pre-warmed at 37°C. The lid was loosened to allow access of CO2 and the cells were incubated at 37°C for at least 2 days with 10% CO2
6. Once a confluent monolayer of cells was present within the flask, the cells were divided as follows: Media was removed and cells were washed once in PBS (ICN Biomedicals Inc.)
2ml of trypsin (lOX in solution, Sigma Chemical Co.) was washed over the monolayer and removed
1 ml of trypsin was added and left in contact with the monolayer for 1 to 2 minutes
cells were loosened by tapping the flask and then washed off with 5ml of pre-warmed media the suspension of cells was divided into the required number of flasks, containing pre-warmed media as before (step 5)
2.2.19 Exon Trapping (Dyuk
et aL,1991, Buckler
etuZ., 1992, Church
et al.,1994)
2.2.19.1 Preparation of pSPL3 vector
1. pSPL3 vector DNA was prepared from a bacterial clone containing the plasmid using a QIAGEN midi-tip according to the manufacturer specification.
2. 5|ig of pSPL3 was completely digested with 50 units of BaniRl restriction enzyme, according to the manufacturer instructions.
3. The digested vector was phenol/chloroform+isoamylalcohol (24:1) extracted, ethanol precipitated (for details see 2.2.12, steps 10-13) and resuspended in 17pl of H^O.
4. IU of alkaline phosphatase along with the appropriate buffer was added to the sample, which was incubated at 37°C for 45 minutes followed by heat inactivation at 68°C for 10 minutes. The DNA was then phenol/chloroform:isoamyalcohol extracted, ethanol precipitated and resuspended in 50pl of H^O.
5. 1 pi of the sample was resolved on a 0.8% agarose gel in the company of a size/weight DNA marker to determine the concentration (DNA was visualized as in section 2.2.3, steps 1+2).
6. Three test ligations were carried out with 25ng of the digested, phosphatased vector. The first contained vector, ligation buffer, H^O and lOmM rATP only. The second contained vector, ligation buffer, H^O, lOmM rATP and lU of ligase. The third reaction contained vector, ligation buffer, H^O, lOmM rATP, lU of ligase and 1U of polynucleotide kinase. Ligations were carried out overnight at room temperature.
7. Competent cells were transformed with the individual ligations. Polynucleotide kinase rephosphorylates the ends of the vector while ligase only ligates phosphorylated ends. Test ligations indicated the presence of undigested or unphosphotased vector.
2.2.19.2 Preparation of bacterial clone insert DNA
1. 15pi of plasmid DNA (PAC) was digested with either SauSAl (partially) or with both BamHl and BgBl
(fully). Five separate 20pl reactions containing the appropriate buffers and 2pg of RNase were set up with 3pl of plasmid in each. For the 5ûm3AI partial digestions 0.5 units of enzyme were added to each of the five
reactions which were incubated for one hour only at 37°C before heat inactivation took place. For the
BamYWIBgUl complete digestions ] Ounitsof each enzyme was added to each reaction and incubated for at least
14 hours at 37®C before heat inactivation.
2. Like reactions were pooled, ethanol precipitated and resuspended in 20|il H^O. 2pl of the BamY{\JBgïïl
digested sample were electrophoresed on a 0.8% agarose gel in order to determine the quality and quantity. All of the 20pl SauiM . partially digested sample was ran on a 0.8% agarose gel in the following way: 3pi of the sample was electrophoresed next to size standards (Ikb, Life Technologies) while the remaining 17pl of the sample was electrophoresed in a neighboring lane of the gel. This was carried out to determine a 3-5kb window by visualizing only the 3pi of sample in order not to expose the remaining 17pl to UV light as this impedes cloning considerably. The 3-5kb window from the portion of the gel containing the 17pi of sample was excised from the gel and purified using the Prep-A-Gene® protocol (see 2.2.3). The purified Sau2)fii\.
sample was resuspended in 30pl of which 3pi was electrophoresed on a 0.8% agarose gel in order to determine quantity and quality.
2.2.19.3 Ligation and transformation
1. Ligation reactions were set up with 30ng of prepared insert DNA {SauiA l partial or BamYWBglll full) and lOng of prepared pSPL3 vector in a lOpl volume of 1 x ligation buffer, lOmM rATP and 0.5 units of ligase. Reactions were left for 14 hours at room temperature.
2. lOOpl of competent cells were transformed with the ligation reactions as described previously except all the transformed cells were plated onto one plate only (see 2.2.17).
3. 2ml of LB containing lOOpg of carbenicylin were added to the agar plates in order to resuspend the colonies which were initially scraped off.
4. The resuspended cells were added to 20ml of LB containing 50pg/ml carbenicyclin and placed in a rotating incubator at 37°C / 200rpm overnight.
5. Plasmid DNA was isolated using a QIAGEN midi-tip (according to the manufacturer specifications) and resuspended in 50pl of H^O. 3pi of the resulting sample was electrophoresed on a 0.8% agarose gel in order to assess quantity and quality.
2.2.19.4 Electroporation
1. A sufficient number of 500ml tissue culture flasks of COS7 cells were cultured to generate one flask per sample plus a positive and negative control. Cells were cultured until they were approximately 75% confluent. Cells were then trypsinated as described and pelleted at 1100 rpm for 5 minutes, washed in PBS (ICN Biomedicals Inc.) and repelleted.
2. Cells were resuspended in 700|il of cold PBS (ICN Biomedicals Inc.) and added to lOOpl + 20|ig of transformant DNA in a pre-cooled 0.4cm cuvettes. The cuvettes were placed on ice for 10 minutes and gently resuspended before electroporation was carried out under the following parameters;
Voltage 1.2kV
Capacitance 25 pF
Resistance lOOQ
Pulse time 0.5-1 second
3. Cells were placed on ice for a further 10 minutes before being transferred to a 500ml tissue culture flask containing 10ml of pre-warmed media. Cells were incubated for 72 hours with a change of media after 24 hours.
2.2.19.5 Reverse transcription and PCR
1. Electroporated cells, grown for 72 hours, were trypsinated and pelleted as described. The supernatant was removed and the pelleted cells resuspended in lOOpl of RNAzol B (Biogene) before being transferred to a
1.5ml eppendorf tube and placed on ice for 1 minute.
2. lOpl of chloroform was added and the reaction was vortexed for 20 seconds and placed on ice for 10 minutes.
3. The reaction was centrifuged at 13,000rpm for 3 minutes after which the aqueous phase was removed to a fresh 1.5ml eppendorf tube.
4. The RNA was precipitated by adding an equal volume of isopropanol and placed at 4°C overnight. 5. The RNA was pelleted at 15,000g / 4°C for 15 minutes. The supernatant was removed and the pellet was washed briefly with 80% v/v ethanol and dried at room temperature for 5 minutes.
6. The pellet was resuspended in 20pl of dEPC treated H^O. 7. RNA was reverse transcribed by first adding:
7pi 5 X RT buffer 4pl lOmM dNTPs
Ipl RNasin (lOU/pl) Ipl primer SA2 (lOOng/pl)
The reaction was incubated at 65°C for 10 minutes and then placed on ice. 2pl of M-MLV reverse transcriptase was added before incubation at 42°C for 90 minutes.
8. Half of the reaction (17.5|il) was removed and added to:
5)il 10 X Guy’s Buffer 1^1 lOmM dNTP’s
2.5^il primer SD6 (lOOng/pl) 2.5pl primer SA2 (lOOng/pl) Ipl Promega Taq
20.5pl HjO
9. Primary PCR was carried out under the following conditions: 6 cycles of: 94“C 1 minute 55®C 1 minute 72°C 5 minutes then held at 55°C
10. 2pi (20 units) of the restriction enzyme BstXl was added and the reaction was incubated at 55°C overnight,
11. A further 0,5pl (5 units) of BstXl was added and incubation was continued for a further 2 hours at 55°C. 12. A secondary, or nested, PCR was set up in the following way:
lOpl primary PCR reaction 5pi 10 X Guy’s Buffer
Ipl lOmM dNTP’s
2.5pl primer SD2 (lOOng/pl) 2.5 pi primer SA4 (lOOng/pl) 29pl H;0
Cycles were carried out as follows: 30 cylces of: 94°C 1 minute 55°C 1 minute 72°C 3 minutes
13. The PCR reaction was ethanol precipitated to remove any excess nucleotides and primers and resuspended in 20pl.
14. 3pi of the secondary PCR was electrophoresed on a 1% agarose gel in the presence of size and weight standards to determine the concentration and quality.
2.2.19.6 Sub-cloning secondary PCR products
1. Secondary PCR products were cloned using the CLONEAMP® pAMPl System (Life Technologies) in the following way:
3|il Secondary PCR product Ipl pAMPl vector
0.5pl 10 X Guy’s Buffer
0.5pi UDG (Uracil DNA Glycosylase)
The reactions were placed at 37°C for 40 minutes before transformation of competent cells (see 2.2.17) which were plated out on LB agar containing 50pg/ml carbenecylin, 20pg/ml IPTG and 20pg/ml X-gal (for blue white screening) and incubated overnight at 37°C.
2. White colonies (insert containing) were used for subsequent analysis.
2.2.19.7 Generation of 3^^ PCR for library screening
1. The following reactions were set up: 36pl H%0
5pi 10 X PCR buffer (supplied with enzyme) Ipl EXl primer
1 pi EX2 primer Ipl lOmM dNTPs
1 pi SUPER TAQ polymerase
3pi secondary PCR product (section 2.2.19.5) Cycles given in section 2.1.4.
2. 10% of the resulting PCR amplification was resolved with 2% agarose gel electrophoresis. 25ng (on average 1 pi of 3"^ PCR) was used in subsequent radio-labeled probe preparation (see 2.2.8).