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The following are the 7 inbred mice strains used in this study. DNA samples of these strains were directly obtained from Dr. Ruth Barber (Department of Genetics, University of Leicester). All the 7 mice strains were obtained from Harlan Laboratories, UK.
The mouse genomic DNA after purification was diluted to 100ng/Âµl and 600ng was digested with 24 units of AseI. The digestion was carried out overnight at 37oC in a 30l reaction volume. Negative controls, Enzyme -ve (enzyme replaced with 50% glycerol), DNA -ve, were set up along with the reaction. After digestion, the enzyme was inactivated by incubation at -15C and then cooled in ice. The reactions were centrifuged briefly and condensate collected and stored at -20oC.100ng of digests was treated with AseI (Over digests) for 3 hours at 37oC. Then 100ng of digests and over digests were fractionated on a 1% agarose gel to check the quality of the digests.
For making the annealed linker molecule, a reaction containing 40l of 20ÂµM oligonucleotide RBMSL2 and 20ÂµM RBD3 each (resuspended in 5mM Tris HCl pH 7.5) was heated at 65oC for 10 minutes. The reaction was slowly brought down to room temperature. Ligation reactions were set up with 100ng (5Âµl) of digested DNA with negative controls, DNA -ve, linker -ve (linker replaced by 50% glycerol) and ligase -ve. Each reaction volume of 20Âµl contained 2.7Âµl of annealed linker, 4 units of T4 DNA ligase with 1X Invitrogen rapid ligation buffer. The ligation was carried overnight at 15C. After the ligation was complete, excess linker from the reaction was removed using a Qiaquick PCR purification kit following manufacturer's protocol.
METHYLATION SENSITIVE VARIANT:
After purification of the ligation reaction, the 30l reaction was split into 3X 10ul aliquots. Each 10ul (~30ng) of DNA was digested in a 20ul reaction with 10 units HpaII, MspI, with appropriate 10X enzyme buffer, and 'mock' digests (enzyme replaced by 50% glycerol). Digestion of all three reactions was carried out at 37oC for at least 3 hours or overnight (14-16 hours). After digestion the enzyme was inactivated by incubating at 65oC for 20 minutes and cooled on ice. 2l of the digested library was seeded into the suppression PCR.
Microamp thin wall PCR tubes (Perkin Elmer) were used to amplify 1l (5ng) of the library. A final reaction volume of 15l contained linker specific primer, RBX4 and 5Â´UTR IAP specific primer, RBIAPUNIB at a concentration 1.2M with 0.05 units/Âµl Taq DNA polymerase (Kappa) and 1X PCR buffer, also called Standard "Jeffreys 11.1X buffer". The cycling conditions are mentioned in appendix. The amplified products were fractionated on a 2% agarose gel by agarose gel electrophoresis. For methylation sensitive variant, concentration and the volume of the reaction components were the same.
A 1 in 10 dilution of the suppression PCR product was obtained by diluting 5Âµl of amplified product with 45Âµl of 5mM Tris, to be seeded in the secondary PCR. Filter tips were used to avoid cross contamination. The final reaction contained 1Âµl of diluted primary PCR in a final volume of 10Âµl using linker specific primer, RRY (RRYATA/ RRYATG/RRYATC/RRYATT) and a nested 5Â´end IAP LTR specific primer, RBIAP5PB2 at a final concentration of 0.625ÂµM. The reaction also contains Taq DNA polymerase whose concentration is 0.05 units/Âµl and Jeffreys 11X buffer. The amplified products were fractionated on a 2% agarose gel by agarose gel electrophoresis.
METHYLATION SENSITIVE VARIANT:
The set of linker specific primer, RRY was replaced by a linker specific primer, RBX4 with no variations in the concentration and volume of other reaction components.
The aim of the PCR was to achieve linear amplification through radioactively labelled display primer. A 20l labelling reaction contained 75pmol of display primer, RBIAPDP1 that was labelled using 50ÂµCi of Î³32P ATP (5l) and 10 units of T4 DNA kinase, Optikinase, in 1X Optikinase buffer. The labelling reaction was incubated at 37 for 60 minutes after which the enzyme was inactivated by heating at 65C for 20 minutes. The reactions were cooled on ice and the condensate collected.
The PCR reactions were setup in the radioactive area, where 1l of the secondary PCR reactions were seeded into a final reaction volume of 10l containing 0.9-1.9pmol of Î³32P ATP labelled primer, 0.02l of Taq DNA polymerase and 1X RMB buffer C. Negative controls were set up from suppression and secondary PCR and the reactions were set up in duplicate. The reactions were amplified using a Perkin Elmer Cetus GeneAmp 9600 PCR machine. The cycle conditions are mentioned in the appendix.
10Î¼l sequencing stop solution was added to each reaction after the cycles. The reactions were then incubated at 96oC for 4 minutes and were snap cooled on ice for 5 minutes. 3l of each reaction was loaded on the denaturing urea-polyacrylamide gel with radiolabelled 100bp molecular weight marker.
Î³32P 100bp Ladder:
A radioactively labelled 100bp DNA molecular weight marker was made by setting up a 10l reaction containing 1l (500g/ml) of 100bp ladder with 2 units of SAP- Shrimp Alkaline Phosphatase (1unit/l, Fermaentas, EU) and 1l 10X SAP buffer (Fermentas, EU) and 6l of ultrapure 18â„¦ millipore water. The reaction was incubated at 37C for an hour and then the enzyme was heat inactivated at 80C for 20 minutes. The reaction was labelled with 1Î¼l Î³32P dCTP using 10 units of Optikinase (10 units/Î¼l from USB) and 2Î¼l of 10X Optikinase buffer and brought up to 20l with ultrapure 18â„¦ millipore water. The final 20l reaction was incubated at 37C for an hour before 20l of sequencing stop solution was added to it.
Denaturing Urea-polyacrylamide Gel
The gel mix for the 6% display was prepared well in advance and stored at 4C. The gel mix for a 6% gel was prepared in advance and stored at 4oC. Gel mix 100mls and 20X Glycerol tolerant Gel Buffer 1L were mixed in two separate beakers using a magnetic stirrer and then filter sterilized using 0.22Âµm Millipore Express Plus membrane filter. 100Âµl of freshly prepared 25% APS and 100Âµl of TEMED were added to the gel mix (~100 ml) before the gel was poured and run according to manufacturer's (Bio-Rad) instructions. The gel was dried under vacuum at 80oC for 2 hours after the run using a Bio-Rad model 583 dryer.
Recovery of Display PCR products
The dried gel was aligned with the developed film or autoradiograph to excise the band of interests from the gel using sterile scalpel blades. After excision, the gel was autoradiographed again to ensure that the right bands were excised. The excised gel fragments were frozen overnight in 20l of 5M Tris-HCl pH 7.5 at -20C. Three 10l PCR reactions were set up for each excised band, one with the display primer, RBIAPDP1 alone, one with linker specific primer, RBX4 alone and the third reaction was amplified with both the primers. A 10l reaction contained primers at a concentration of 0.625Î¼M, along with 0.2 units of Taq DNA polymerase and 1X Jeffreys buffer with 1l of thawed eluate. The amplified products were fractioned on a 2% agarose gel. The amplified products were purified using a Zymoclean Gel DNA Recovery Kit (Zymo research) according to the manufacturer's instructions.
A 10l sequencing reaction was set up for all the display products that were exponentially amplified (with both primers), containing 20-30ng/Kb of template DNA, 1.5l of 5X Big Dye Terminator Buffer with 1l of Big Dye Terminator ready reaction mix and 1l of 3.2M sequencing primer. The sequencing reactions were cycled using a MJ DNA tetrad PCR machine PCT250 with the cycle conditions mentioned.
Clean up of Sequencing Reaction
To the 10l of the sequencing reaction, 10l of ultra pure 18â„¦ millipore water and 2l of 2.2% SDS were added and mixed thoroughly. The reaction was incubated at 98C for 5 minutes and then incubated at 25C for 10 minutes. Edge Biosystems Performa DTR gel filtration column were used to remove the excess dye terminators. The columns were spun first at 3200rpm for 3 minutes to pack it and then spun again at 3200rpm for 3 minutes with sample loaded. The eluate was submitted to PNACL for sequencing.
Ultra Competent E.coli cells were prepared following the protocol by Inoue H., et al. in 1990. pGEM-T easy Kit (Promega) for cloning, according to manufacturer's instruction.
Transformation was carried out by adding 5l of the ligation mix to 200l competent cells and left on ice for 30 minutes, flicking gently occasionally, followed by heat shock in heat block at 42C for 30 seconds, and snap cooled on ice for 2 minutes. The transformed cells were plated on LB ampicillin plates (0.2mg/ml Ampicillin and Luria Broth) containing 40Î¼l of 50mg/ml Xgal and 20Î¼l of 24mg/ml IPTG. This facilitates the Blue/White screening. The plates were incubated overnight at 37C. Qiagen miniprep kit (Crawley, UK) was used for plasmid extraction, following the manufacturer's instructions.
Genotyping PCRs were carried out in a MJ DNA tetrad PCR machine PCT250. 1000ng of genomic DNA was digested with 10 units of Hpa II and Msp I along with mock digests (containing 50% glycerol in the place of restriction enzyme). 1l of the digested DNA was amplified in a 20l reaction containing primers at a concentration of 0.625M, with 0.2 units of Taq DNA polymerase and 1l of 10X RMB buffer B, and the volume made up to 20l using PCR clean 18Mâ„¦ ultrapure water. The genotyping primers and the cycling conditions are mentioned in the appendix.
Agarose gel electrophoresis
LE (Low Electroendosmosis) Agarose
The PCR products were fractionated on LE agarose gel (SeaKem) using 0.5XTBE buffer containing 0.5g/ml of Ethidium Bromide. 100bp (NEB) and 1Kb (NEB) molecular weight markers were used to determine the size of the fractionated products on the gel. 2% w/v agarose gels were used for products less than 1Kb while 1%w/v gels were used for 1-2Kb products. The samples were loaded on the gel with the loading dye and were run for 1-2hrs at 100-120V depending on the size of the fractionated products. After the run, the fragments were visualized using UV transilluminator (Clare Chemical Research), and a CCD camera equipped UV transilluminator cabinet (Syngene) and GeneSnap software (Syngene) were used to capture the gel images. The power packs for the electrophoresis were from Bio-Rad.
LMT (Low Melting Temperature) Agarose
LMT agarose gels were used for extraction of DNA for purification, using 0.5X TBE buffer with 0.5g/ml of Ethidium Bromide. The gel was poured at 4C in the gel casting tray to set. The electrophoresis characteristics of LMT gels are similar to those of conventional agarose gels. The fragments after fractionation were excised and dissolved in 5 volumes of 20mM Tris HCl (pH 8.0) and 1mM EDTA and heated for 5 minutes at 65C. The melted gel slice was extracted by standard Phenol-Chloroform extraction and ethanol precipitation.
The DNA from the gel was transferred to a nylon membrane by following standard Southern Blotting protocol with a few minor modifications. The molecular weight markers on the gel were marked by making nicks and after blotting, they were marked on the blot to identify the size of the products after hybridization.
Single stranded oligonucleotide probes
Î³32P dATPs were used to label these probes using Optikinase enzyme (USB). A 10l labelling reaction contained 1l of 2pM of oligonucleotide labelled with 0.5Î¼l of Î³32P dATPs using 5 units of Optikinase enzyme and 1X Optikinase buffer (USB), the volume made up to 10l with ultra pure 18Mâ„¦ ultrapure water, and the reaction was incubated at 37C.
Prehybridization of the membrane was carried out in 30-50mls of CHURCH solution at 55C for 60 minutes. The labelled probe was added to the hybridization bottle with the membrane after replacing the prehybridization solution by a fresh 30-50mls of CHURCH. The hybridization was carried out at 55C for 3-4 hours.
After hybridization, a first wash of the membrane was carried out by replacing the solution in the bottle by a fresh 30-50mls of CHURCH solution and incubated at 55C for 15 minutes, followed by a second wash with the same amount of CHURCH at 55C for 15 minutes. The membrane was wrapped in Saran wrap and was exposed to an X-ray film in a cassette, overnight.
Double Stranded DNA probes
Standard double stranded DNA probe labelling protocol was used for genotyping assays. A probe recovery was done prior to hybridization and the hybridization was carried out overnight. The membrane was washed and autoradiographed overnight.
Jeffreys 11.1X Buffer
Non radioactive PCR assays
50mM Tris HCl, 12mM NH4SO4, 5mM MgCl2, 7.4 2-mercaptoethanol, 125 Âµg/ml BSA, 1.1mM dNTPs
1X RMB buffer C
(10 mM Tris HCl pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 250 mM dNTPs)
Sequencing Stop Solution
95% Formamide, 20mM EDTA, 0.05% Bromophenol Blue, 0.05 % Xylene Cyanol FF
Denaturing Urea Polyacrylamide gel mix 100ml
50g Urea, 12ml Long Ranger gel solution from Lonza, 4ml 20 X Glycerol tolerant buffers, and volume made up to 100ml with ultra pure 18â„¦ milipore water
20X Glycerol tolerant Gel Buffer 1L
216g Tris Base, 72g Taurine, 4g EDTA and ultra pure 18â„¦ milipore water ~800mls
2ml 0.5M EDTA, 500ml 1M NaHPO4 (pH 7.2), 500ml 14% SDS