BAC/PAC 克隆完全攻略,强烈申请加分!!
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一.Recommendations on how to handle
the clones you receive
The clones you receive from us are shipped as bacterial LB agar stab culture.The DH10 E.coli host that is harboring the plasmid contains a DNA insert that has been placed into LB agar (containing 20 ug/ml chloramphenicol for BAC clones and 25 ug/ml kanamycin for PAC clones). This culture has a finite life at 4°C. We recommend that you streak this culture to single colonies on a LB agar plate with an antibiotic and prepare a glycerol stock of the clones for long term storage at -85°C. While our libraries have little well to well contamination it would be wise, before initiating any experiments, to check 3-5 colonies on CHEF gel or PCR. This to make sure that the clone stock that we sent you is pure.
二. Hybridization of High Density Filters
Hybridization of filters can be performed in either rotisserie bottles or heat-seal bags. In our lab both work well. We routinely hybridize up to seven filters in one bag and up to five filters in a large (7.5cm diameter) bottle (with nylon mesh between filters). The filters have been successfully hybridized using a number of different published "Southern" DNA hybridization protocols incorporating a variety of buffer systems. It has been our experience that all of these established protocols will work with the high density filters. In our lab we routinely follow the following procedure:
Wet filters in minimum volume of "Church Buffer" in large petri dish. Keep track of volume of buffer absorbed into the filters as this will contribute to your overall volume. If using bottles, place nylon mesh between filters as you wet them.
Place filters in bottle or bag, add approx. 25ml additional "Church Buffer" and seal.
Pre-hybridize one hour at 65?.
Add labeled probe(s) so that the total cpm of each probe is 1X106 to 11X107. We routinely label our probes by random prime labeling. PCR incorporation also works well for smaller (>100-200bp) probes.
Hybridize overnight (16-18 hours) at 65o C.
Wash filters (in bag or bottle) with Wash I solution at 65o C. Repeat wash about 4 to 6 times until most of the non-bound probe is removed. At this point the filters can continue to be washed in the bag or bottle or placed in a large tray in a 65o C water bath for more efficient washing.
Wash filters with wash II solution repeatedly at 65o C until the level of cpm (monitored with a Geiger counter) removed remains constant (no longer decreases).
Rinse filters in water and wrap individually in plastic wrap. Take care to remove air bubbles between filter and wrap by pressing with a wipe to force out air.
Place two filters in a large cassette (35x43cm), there will be a small overlap of filters but this normally is not a problem in interpreting data. We place two filters per cassette to maximize film use.
Expose for 2 to 72 hours at -80o C. This depends on the intensity of signal from filters. We have found it best to develop one piece of film after 4-5 hours exposure to determine the optimum exposure time required for all of the filters.
Interpret the position of the positive clones following the protocol supplied with the filters using the transparent overlay as an orientation guide.
Store the used filters in a sealed hybridization or zip-lock bag in "Church Buffer" to keep them wet. Allow the radioactivity to decay out for 2-4 half lives before using again. We do not recommend stripping the filters as smearing of the DNA may result and the overall life of the filters may also be diminished.
Blocking:
We block our nonspecific binding of probes by adding an equal volume of sonicated human placental DNA (10mg/ml) to our probe solution, incubate one hour at 65o C before adding the probe to the hybridization bag/bottle. Alternatively, the sonicated human placental DNA can be added during the pre-hybridization to the bag/bottle at a level of 100-200 ug/ml of hybridization solution.
Solutions:
Church Buffer -- 1 Liter BSA 10 gm
0.5M EDTA 2 ml
1 M NaHPO4(pH: 7.2) 500 ml
20% SDS 350 ML
H20 to 1 liter
--------------------------------------------------------------------------------
Wash I -- 2 Liters BSA 10 gm
0.5M EDTA 2 ml
1 M NaHPO4(pH: 7.2) 80 ml
20% SDS 500 ml
H20 to 2 liters
--------------------------------------------------------------------------------
Wash II -- 4 liters 0.5M EDTA 8 ml
1 M NaHPO4(pH: 7.2) 160 ml
20 % SDS 200 ml
H20 to 4 liters
--------------------------------------------------------------------------------
1 M NaHPO4(pH: 7.2) -- 1 liter Na2HPO4-7H2O 134 gm
85% H3PO4 4 ml
三.
DNA Isolation From BAC & PAC Clones
This is a rapid alkaline lysis miniprep method for isolating DNA from large PAC clones. It is a modification of a standard Qiagen-Tip method that uses no organic extractions or columns. The method works very well for doing analytical restriction digests of PAC clones and can be scaled up if necessary.
Solutions
P1 (filter sterilized, 4oC)
50mM Tris, pH 8
10 mM EDTA
100 ug/ml RNase A
P2 (filter sterilized, room temp)
0.2N NaOH
1% SDS
P3 (autoclaved, 4oC)
3M KOAc, pH 5.5
Method
1. Using a sterile toothpick, inoculate a single isolated bacterial colony into 2 ml TB (or LB) media supplemented with 25 ug/ml kanamycin. Use a 12-15 ml snap-cap polypropylene tube. Grow overnight (up to 16 h) shaking at 225-300 rpm at 37?C.
2. Remove toothpicks using forceps. Centrifuge (SM24 or similar rotor) at 3,000 rpm for
10 min. of spin in the Sorvall. The temperature ot the spin is not critical at this stage.
3. Discard supernatants. Resuspend (vortex) each pellet in 0.3 ml P1 solution. Add 0.3 ml of P2 solution and gently shake tube to mix the contents. Let sit at room temperature for 5 min or so. The appearance of the suspension should change from very turbid to almost translucent.
4. Slowly add 0.3 ml P3 solution to each tube and gently shake during addition. A thick
white precipitate of protein and E. coli DNA will form. After adding P3 solution to every
tube, place the tubes on ice for at least 5 min.
5. Place tubes in the SM24 rotor and spin at 10,000 rpm for 10 min at 4 oC.
6. Remove tubes from centrifuge and place on ice. Transfer supernatant using a P1000
or a disposable pipette to a 1.5 ml eppendorf tube that contains 0.8 ml ice-cold isopropanol. Try to avoid any white precipitate material. Mix by inverting tube a few times; place tubes on ice for at least 5 min. At this stage, samples can be left at -20? C
overnight.
7. Spin in cold microfuge for 15 min.
8. Remove supernatant and add 0.5 ml of 70% EtOH to each tube. Invert tubes several times to wash the DNA pellets. Spin in cold microfuge for 5 min. Optional:repeat step 8.
9. Remove as much of the supernatant as possible. Occasionally, pellets will become dislodged from the tube so it is better to carefully aspirate off the supernatant rather than pour it off.
10. Air dry pellets at room temp. When the DNA pellets turn from while to translucent in appearance, i.e., when most of the ethanol has evaporated, resuspend each in 40 ul TE. Do not use a narrow bore pipets tip to mechanically resuspend DNA sample; rather, allow the solution to sit in the tube with occasional tapping of the bottom of the tube. For large PAC clones resuspension may take over 1 hour.
11. Use 5 ul for digestion with Notl or other rare cutter enzymes. There are Notl sites flanking the Sp6 and T7 promotor regions of the CYPAC2 vector; therefore, this is a very useful enzyme for analysis of insert size and for partial digest restriction mapping. Use 7-10 ul for digestion with a more frequent cutter such as BamHI or EcoRI.
the clones you receive
The clones you receive from us are shipped as bacterial LB agar stab culture.The DH10 E.coli host that is harboring the plasmid contains a DNA insert that has been placed into LB agar (containing 20 ug/ml chloramphenicol for BAC clones and 25 ug/ml kanamycin for PAC clones). This culture has a finite life at 4°C. We recommend that you streak this culture to single colonies on a LB agar plate with an antibiotic and prepare a glycerol stock of the clones for long term storage at -85°C. While our libraries have little well to well contamination it would be wise, before initiating any experiments, to check 3-5 colonies on CHEF gel or PCR. This to make sure that the clone stock that we sent you is pure.
二. Hybridization of High Density Filters
Hybridization of filters can be performed in either rotisserie bottles or heat-seal bags. In our lab both work well. We routinely hybridize up to seven filters in one bag and up to five filters in a large (7.5cm diameter) bottle (with nylon mesh between filters). The filters have been successfully hybridized using a number of different published "Southern" DNA hybridization protocols incorporating a variety of buffer systems. It has been our experience that all of these established protocols will work with the high density filters. In our lab we routinely follow the following procedure:
Wet filters in minimum volume of "Church Buffer" in large petri dish. Keep track of volume of buffer absorbed into the filters as this will contribute to your overall volume. If using bottles, place nylon mesh between filters as you wet them.
Place filters in bottle or bag, add approx. 25ml additional "Church Buffer" and seal.
Pre-hybridize one hour at 65?.
Add labeled probe(s) so that the total cpm of each probe is 1X106 to 11X107. We routinely label our probes by random prime labeling. PCR incorporation also works well for smaller (>100-200bp) probes.
Hybridize overnight (16-18 hours) at 65o C.
Wash filters (in bag or bottle) with Wash I solution at 65o C. Repeat wash about 4 to 6 times until most of the non-bound probe is removed. At this point the filters can continue to be washed in the bag or bottle or placed in a large tray in a 65o C water bath for more efficient washing.
Wash filters with wash II solution repeatedly at 65o C until the level of cpm (monitored with a Geiger counter) removed remains constant (no longer decreases).
Rinse filters in water and wrap individually in plastic wrap. Take care to remove air bubbles between filter and wrap by pressing with a wipe to force out air.
Place two filters in a large cassette (35x43cm), there will be a small overlap of filters but this normally is not a problem in interpreting data. We place two filters per cassette to maximize film use.
Expose for 2 to 72 hours at -80o C. This depends on the intensity of signal from filters. We have found it best to develop one piece of film after 4-5 hours exposure to determine the optimum exposure time required for all of the filters.
Interpret the position of the positive clones following the protocol supplied with the filters using the transparent overlay as an orientation guide.
Store the used filters in a sealed hybridization or zip-lock bag in "Church Buffer" to keep them wet. Allow the radioactivity to decay out for 2-4 half lives before using again. We do not recommend stripping the filters as smearing of the DNA may result and the overall life of the filters may also be diminished.
Blocking:
We block our nonspecific binding of probes by adding an equal volume of sonicated human placental DNA (10mg/ml) to our probe solution, incubate one hour at 65o C before adding the probe to the hybridization bag/bottle. Alternatively, the sonicated human placental DNA can be added during the pre-hybridization to the bag/bottle at a level of 100-200 ug/ml of hybridization solution.
Solutions:
Church Buffer -- 1 Liter BSA 10 gm
0.5M EDTA 2 ml
1 M NaHPO4(pH: 7.2) 500 ml
20% SDS 350 ML
H20 to 1 liter
--------------------------------------------------------------------------------
Wash I -- 2 Liters BSA 10 gm
0.5M EDTA 2 ml
1 M NaHPO4(pH: 7.2) 80 ml
20% SDS 500 ml
H20 to 2 liters
--------------------------------------------------------------------------------
Wash II -- 4 liters 0.5M EDTA 8 ml
1 M NaHPO4(pH: 7.2) 160 ml
20 % SDS 200 ml
H20 to 4 liters
--------------------------------------------------------------------------------
1 M NaHPO4(pH: 7.2) -- 1 liter Na2HPO4-7H2O 134 gm
85% H3PO4 4 ml
三.
DNA Isolation From BAC & PAC Clones
This is a rapid alkaline lysis miniprep method for isolating DNA from large PAC clones. It is a modification of a standard Qiagen-Tip method that uses no organic extractions or columns. The method works very well for doing analytical restriction digests of PAC clones and can be scaled up if necessary.
Solutions
P1 (filter sterilized, 4oC)
50mM Tris, pH 8
10 mM EDTA
100 ug/ml RNase A
P2 (filter sterilized, room temp)
0.2N NaOH
1% SDS
P3 (autoclaved, 4oC)
3M KOAc, pH 5.5
Method
1. Using a sterile toothpick, inoculate a single isolated bacterial colony into 2 ml TB (or LB) media supplemented with 25 ug/ml kanamycin. Use a 12-15 ml snap-cap polypropylene tube. Grow overnight (up to 16 h) shaking at 225-300 rpm at 37?C.
2. Remove toothpicks using forceps. Centrifuge (SM24 or similar rotor) at 3,000 rpm for
10 min. of spin in the Sorvall. The temperature ot the spin is not critical at this stage.
3. Discard supernatants. Resuspend (vortex) each pellet in 0.3 ml P1 solution. Add 0.3 ml of P2 solution and gently shake tube to mix the contents. Let sit at room temperature for 5 min or so. The appearance of the suspension should change from very turbid to almost translucent.
4. Slowly add 0.3 ml P3 solution to each tube and gently shake during addition. A thick
white precipitate of protein and E. coli DNA will form. After adding P3 solution to every
tube, place the tubes on ice for at least 5 min.
5. Place tubes in the SM24 rotor and spin at 10,000 rpm for 10 min at 4 oC.
6. Remove tubes from centrifuge and place on ice. Transfer supernatant using a P1000
or a disposable pipette to a 1.5 ml eppendorf tube that contains 0.8 ml ice-cold isopropanol. Try to avoid any white precipitate material. Mix by inverting tube a few times; place tubes on ice for at least 5 min. At this stage, samples can be left at -20? C
overnight.
7. Spin in cold microfuge for 15 min.
8. Remove supernatant and add 0.5 ml of 70% EtOH to each tube. Invert tubes several times to wash the DNA pellets. Spin in cold microfuge for 5 min. Optional:repeat step 8.
9. Remove as much of the supernatant as possible. Occasionally, pellets will become dislodged from the tube so it is better to carefully aspirate off the supernatant rather than pour it off.
10. Air dry pellets at room temp. When the DNA pellets turn from while to translucent in appearance, i.e., when most of the ethanol has evaporated, resuspend each in 40 ul TE. Do not use a narrow bore pipets tip to mechanically resuspend DNA sample; rather, allow the solution to sit in the tube with occasional tapping of the bottom of the tube. For large PAC clones resuspension may take over 1 hour.
11. Use 5 ul for digestion with Notl or other rare cutter enzymes. There are Notl sites flanking the Sp6 and T7 promotor regions of the CYPAC2 vector; therefore, this is a very useful enzyme for analysis of insert size and for partial digest restriction mapping. Use 7-10 ul for digestion with a more frequent cutter such as BamHI or EcoRI.
