In theory, once the cosmid DNA has been inserted into the M13 vector, the samples can be sequenced. Each plaque on the plate represents a unique insert. In actuality, there exists the possibility of having various contaminants. For instance, since the cosmid is randomly sonicated into various fragments, it is possible that an inserted fragment could possess nothing but cosmid sequence. Since this is of no interest or particular value to us, sequencing this plaque/sample would waste time and re sources. The chemiluminescent hybridization procedure has been devel oped to help screen out these irrelevant sequences. Using labeled DNA, it is possible to probe unknown samples to determine whether or not the samples are useful. An antibody will recognize and bind to the label, and when this antibody is exposed to certain chemicals it will react and give off light. By probing a series of 96-well plates it is possible to sequence only samples with yeast inserts. The current hybridization protocol uti lizes two probes. The first probe is with the DNA from which the acutal library was made. This would screen out things such as M13 without inserts that were accidentally picked. The second probe is the vector DNA from which the source DNA was created without the in sert. For instance, this would be cosmid without insert or lambda with out insert. This screens out M13 with predominantly useless sequences. Thus, in the first probe, anything that does not have the sonicated DNA will be screened out, and the second probe will screen out those soni cated DNA which does not contain novel yeast sequence. This protocol's utility because a third probe can be introduced. The initial set of cosmids were chosen because they minimized the amount of overlap from one cosmid to another. However, some cosmids do not grow. The alternate cosmids may overlap extensively with already sequenced. To sequence overlapping regions is a waste of time and resources. By using a cosmid as a third probe one can eliminate the overlapping region.
NOTE: Never touch the membrane with your bare hands. Al ways wear gloves! throughout the procedure!!!
Blotting of DNA on Biodyne Membrane by the Biomek:
1-Cut a piece of Hybond-N membrane to a size that corresponds to the size of the Cetus plate base.
Biodyne is a nylon membrane which will bind to DNA.
2-Place the membrane on the base plate and place the plate into the T2 posi tion of the Biomek.
3-In the T1 position place the water-ethanol reservoir and fill it appropriately
with dH2O & ethanol.
4-Under the tool rack next to the water ethanol reservoir place the bleach reservoir and fill it.
The bleach wiill be used to clean the probe as it takes from various samples.
5-Place the 96-pin tool above the bleach on the first tool rack.
6-On the tool rack next to T3 place the fan.
7-Place the 96 deep-well plates on the four-shelf racks starting at position A1.
8-Log onto Marmite from any alpha terminal; type " guile".
9-Choose the pinblot protocol.
10-Enter the number of plates to be blotted and click " continue".
11-Follow instructions of the Biomek program as seen on screen.
12-Make sure the pins are touching the liquid and the membrane.
Blotting the Membrane with Control DNA
1-Make the Negative and Positive Control solutions
Negative Control
Mix by vortexing or pipetting.
Positive Control
Mix by vortexing or pipetting.
2-If available make a second positive control from unlabeled probe DNA.
2nd Positive Control
Mix by vortexing or pipetting.
DNA #4 is a labeled strand of DNA that will control for the effectiveness of the detection system.
DNA #4 and M13 are single stranded and do not need to be denatured.
All other controls must be denatured by boiling on a 95 oC block for 10 min and then placing on ice for 3 min.
3-Carefully place ~ 0.3 µl of each control on the left side of the membrane away from the plate blots. Mark the upper left-hand corner of the membrane with a scissor cut. This indicates which side lies the DNA.
Locations of Controls on Membrane:
4-Place the membrane face-up into the strategene UV stratalinker.
5-Turn on power, press "Autocrosslink ", and press start.
This will fix the DNA to the membrane.
6-Wrap the membrane around a 15-ml Falcon tube and place both inside a 50 -ml Falcon tube .
Hybridization Incubation
1-Pour 30 ml of hybridization solution into the 50-ml tube with the membrane.
The hybridization solution is also known as the blocking solution. Essen tially, the proteins in the blocking solution will bind with any portions of the membrance that do not already have DNA on it. Without the block ing solution, the primary probe would bind to the membrane non-specifi cally which would lead to a high background.
20X SSC
Hybridization Solution
2-Place membrane in the 68°C air oven for 1 hr.
3-Place concentrated probe on a 95°C block for 10 min. Then place it on ice for 3 min.
The high temperature will cause the probe to denature into a single -stranded form.
4-Pour out the Hybridization Solution and add 15 ml of Probe Solution.
The Probe Solution is made by adding probe (usu. 10 µl) to 15 ml of Hybridization Solution. The amount of probe added depends on the con centration of the probe, although 10 µl is usually adequate.
5-Place the membrane (still inside the Falcon tube) back into the 68 oC air oven.
6-Incubate at least 20 hr or 2 days for best results.
The signal increases with longer incubation times.
Wash
1-Pour out the Probe Solution in to another Falcon tube and store at -20 C.
The probe solution is reused for subsequent hybridizations by adding another 10 µl of probe.
2-Pour 20-30 ml of Wash Solution #1 into the Falcon tube containing the membrane.
Wash Solution #1
3-Place the tube on rotator for 5 min at room temperature.
4-Remove wash solution. Repeat wash.
5-Remove wash solution.
7-Pour 30 ml of Wash Solution # 2 into the tube.
Wash Solution #2
8-Place container in pre-heated 68oC air oven for at least 15 minutes.
9-If the probe was weak, skip second wash; otherwise, wash again.
10-Remove wash solution #2 from container.
Blocking
1-Add 30 ml of Blocking Solution to the tube containing the membrane.
10% Blocking Reagent
Blocking Solution
Mix by vortexing
2-Incubate 30 min at room temperature on the rotator.
Substrate Solution
Use a 1 ml syringe and needle to withdraw CPSD from bottle. Dis pense CPSD into substrate buffer. Rinse needle in buffer. Mix by vortexing. CSPD must be stored at 4°C, in the dark.
Note: Dispose of syringe and needle in sharps container.
Antibody Incubation
1-Prepare Antibody Solution.
Buffer #1 Solution
Antibody Solution
Keep the Antibody and solutions cold as it will degrade at room temperature. Mix solutions by vortexing.
2-Remove blocking solution. Add antibody solution to the membrane and incu bate on rotator for 30 min to 1 hr at room temperature.
3-Remove antibody solution. Incubate membrane in buffer #1 on rotator for 5 minutes. Remove solution
4-Repeat wash step 3 times. Discard buffer #1.
Substrate Incubation
1-Add substrate solution. Incubate on shaker for 5-15 min at room tempera ture.
Substrate Buffer
CPSD is a compound which will be altered by alkaline phosphatase. When activated, it will emit light which can then be utilized to develop film.
2-Place membrane face-down on a piece of saran wrap and cover both sides of membrane.
When handling the membrane, only handle the four corners. Make sure that the Saran Wrap is large enough to cover both sides of the mem brane.
3-Fold the saran wrap around the membrane.
Make sure that the Saran Wrap comes together at the back of the membrane.
4-Place membrane face-up inside a film cassette.
5-Incubate cassette at 37°C for 45-60 min. The membrane cannot be left longer than three hours.
Film Exposure
1-In the dark room, place a piece of film on top of the membrane, close the cassette tightly and let it expose the film for approximately 20 - 30 min.
Optimum exposure time may vary widely i.e., 1 - 2 hours to overnight.
The membrane may be stored at 4°C overnight ir you wish to expose it to film on the next day.
2-Turn on developer.
3-Remove film from cassette (in the dark!) and put it through the developer.
If the signal of the film is too faint, try a longer exposure time.