10. Add 50 µl of 45% (w/v) sterile glycerol to each well of any working plates that are to be frozen (-80°C) and subsequently used as culture sources.
2.2 Isolate plasmid templates
1. Warm the lysis buffer (Edge Biosystems Kit) to 37°C to dissolve the SDS. This buffer can be stored at room temperature.
2. Add the RNAse solution to the resuspension buffer (Edge Biosystems Kit), 1ml/100ml. Store at 4°C. The remaining reagents from the kit, neutralization buffer and precipitation buffers, are ready to use and should be stored at 4°C.
3. Prepare the receiving plates from the Edge Biosystems Kit by adding 350 µl of ethyl alcohol to each well of the receiving plates. Place the filter plate on top and secure in place with tape. Handle with care as the wells will be very full.
4. Centrifuge the bacterial cultures in the deep well plates at 1500 x g for seven minutes in a centrifuge equipped with a horizontal rotor for 96-well plates.
5. Invert briefly and tap out excess media on a clean paper towel. Do not delay or the pellets will loosen and may be lost when pouring off excess media.
6. Resuspend the pellet in 100 µl of Resuspension Buffer. Vortex until entire pellet is re-suspended. This step is critical. Poor resuspension of the cells results in clumps of cells that do not lyse in subsequent steps. This reduces the yield and decreases the purity of the product.
7. Add 100 µl of Lysis Buffer. Mix gently by rocking the plates from side, avoid shearing the bacterial chromosomal DNA.
8. Add 100 µl Precipitation buffer to each well. Mix briefly.
9. Add 100 µl Neutralization buffer to each well. Vortex.
10. Transfer the contents of the deep wells to the waiting filter plates/receiving plate stacks using the wide bore pipette tips provided in the kits.
11. Centrifuge the stacked plates at 1500 x g for twelve minutes in a centrifuge equipped with a horizontal rotor for 96-well plates.
12. Remove the stacked plates from the centrifuge. Remove and discard the filter plates . Decant the alcohol and filtrate from the receiver plate. Touch off excess alcohol on clean paper towels.
12. Add 500 µl of 70% ethanol to each well. Decant immediately. Touch off excess alcohol on clean paper towels.
14. Place plates in a clean drawer without their lids, cover with a clean paper towel and allow to dry overnight.
15. Re-suspend DNA in 200 µl of T Low E Buffer. Seal top with plate sealer. Rehydrate at 4°C for at least two days before using. Store at -20°C.
2.3 Amplify EST inserts
1. For each 96 well plate to be amplified, prepare a PCR reaction mixture containing the following ingredients:
1000 µl
10X PCR Buffer
20 µl
dATP (100 mM)
20 µl
dGTP (100 mM)
20 µl
dCTP (100 mM)
20 µl
dTTP (100 mM)
5 µl
AEK M13F primer (1 mM)
5 µl
AEK M13R primer (1 mM)
100 µl
Ampli-Taq polymerase (5 U/µl)
8800 µl
H2O
2. Label 96-well PCR plates and aliquot 100 µl of PCR reaction mix to each well. Gently tap plates to insure that no air bubbles are trapped at the bottom of the wells.
3. Add 1 µl of purified EST plasmid template to each well.
Mark the donor and recipient plates at the corner near the A1 well to facilitate correct orientation during transfer of the template. It is important to watch that the pipette tips are all submerged in the PCR reaction mix when delivering the template. Missing the liquid is easier when multi-channel pipettes are used.
上一篇：Genomic DNA Labeling Protocol 下一篇：E.coli Total RNA Labeling Protocol for Spotted Microarray
共12页: 上一页 [1] [2] 3 [4] [5] [6] [7] [8] [9] [10] [11] [12] 下一页
