The overall goal of this procedure is to investigate the DNA damage checkpoint using xip egg extracts as a model system. First, prepare low speed extract from xip eggs. Then prepare damaged sperm chromatin with DNA damaging approaches such as MMS and UV light.
Also prepare the DNA damage mimicking structure. A T 70 proceed to trigger the DNA damage checkpoint in LSE with the damaged sperm chromatin or the a T 70. Ultimately, results from blots can provide insights into the a TR check one mediated DNA damage checkpoint in the xenopus egg extract.
Hello there. This is CH Y from the department biology at UNC Charlotte. Today we will show you how pus extract is used as model system to investigate the damage tech point signaling.
This method can help answer key questions in the dynamic instability field, such as how D damage checkpoint is activated. This protocol is for the preparation of both opus egg extract and DNA damage checkpoint inducing reagents and the analysis of DNA damage checkpoint and xap. These techniques are adaptable to a variety of DNA damaging approaches, including four NQO and etoposide.
To study the DNA damage checkpoint, let's get started. To prime the female xap lay of his frogs subcutaneously, inject the dorsal lymph sacs with 100 units of PMSG. After waiting a minimum of two days after priming induce egg laying by, injecting each primed frog with 500 units of human chorionic gonadotropin subcutaneously in the dorsal limp sax, incubate the injected frogs for 14 to 20 hours in separate buckets containing two liters of Marx modified ringer solution.
Remove the frogs from the buckets and pour off the MMR solution until around 100 milliliters is left. Then transfer the eggs from the buckets to a 250 milliliter beaker de jelly the eggs by adding 100 milliliters of 2%cysteine pH 7.8 approximately every 30 seconds. Gently swirl the eggs with an inverted glass pasture pipette of 0.7 centimeters in diameter, decant and replace with fresh cysteine twice.
The de detailing process will be complete in about five to 15 minutes. When the eggs form a more condensed layer at the bottom of the beaker After discarding the cysteine solution, wash the eggs three times with 0.25 XMMR solution. Swirl the eggs with a glass pipette and inspect for puffy white bad eggs that tend to accumulate at the center of the beaker.
Remove the bad eggs with a pasture pipette. Next, wash the eggs three times with egg lysis buffer. Remove any additional bad eggs, then pour the eggs into a 14 milliliter falcon tube.
Compact the eggs by centrifugation for 55 seconds at 188 times G.Using a clinical tabletop centrifuge with a swinging bucket rotor, remove the excess puffer above the egg layer. Now for each milliliter of compacted eggs, add 0.5 microliters of 10 milligrams per milliliter of aprotinin, leptin or AL and 0.5 microliters of five milligrams per milliliter of cytokine B or cyto B centrifuge the eggs at 16, 500 times G for 15 minutes at four degrees Celsius. Using the Sarva RC six plus super speed centrifuge with an HB six swinging bucket Rotor after centrifugation ex are fractionated into three layers in the tube lipid extract and yolk pigment from the top to bottom respectively puncture the side of the falcon tube in the lower portion of the middle extract layer with a 21 gauge needle.
Carefully remove the needle as the puncturing needle may be obstructed by plastic. Insert a fresh 21 gauge needle attached to a one milliliter syringe into the puncture site. To collect the extract.
Slowly aspirate the extract, avoiding air bubbles and contamination with the lipid and yolk pigment layers. Transfer the extracts into a chilled 1.5 milliliter micro centrifuge tube. For each milliliter of egg extract, add 10 microliters of 10 milligrams per milliliter.
Cycloheximide one microliter of 10 milligrams per milliliter. A pronin leptin, one microliter of five milligrams per milliliter cytokine B one microliter of one molar DTT and 0.33 microliters of 10 milligrams per milliliter. Ole mixed by inverting the egg extract at least 10 times gently, But low speed extract must be used within four hours as the quality of LSE is compromised.
After four hours or a free thaw Dissolve the two synthetic oligonucleotides in water concentration of two micrograms per microliter. Add 100 microliters of each oligo to a 1.5 milliliter micro centrifuge tube and incubate for five minutes in a 95 degree Celsius heat block. Remove the dry bath block with the tube containing the at oligo mixture and set to cool to room temperature portion 10 microliter aliquots to store at minus 20 degrees Celsius.
The AL sample is now an A T 70 duplex at a final concentration of two micrograms per microliter. Prepare normal sperm chromatin as described by Tudor and Walter to 0.5 milliliters of chromatin. Add 5.5 microliters of 9.1 molar MMS.
Place the sample tube on a rotator at room temperature for 30 minutes using the IE CCL two clinical centrifuge with swinging bucket rotor and tube adapters. Spin the sample at 686 times G for 10 minutes at room temperature. Discard the supernatant and wash the pellet in 0.5 milliliters of buffer X containing B-S-A-D-T-T-A proin and leptin.
Repeat this process three times and resuspend the pellet in 0.5 milliliters. Buffer x. Determine the concentration of sperm chromatin with a hemo cytometer.
Then dilute the sample to 100, 000 sperm per microliter and store five microliter aliquots at minus 80 degrees Celsius. Add desired amount of normal sperm chromatin on the surface of a piece of param and place the sample into a UV crosslinker. Set the desired energy parameter and start to damage the sperm chromatin via UV light.
Use the UV treated sperm chromatin immediately To induce DNA damage checkpoint in the prepared low speed extract. Combine 50 microliters of LSE one microliter of energy mixture and two microliters of damaged sperm chromatin. Incubate the reaction at B room temperature for 90 minutes, making sure to flick the tube every 10 minutes after at least 30 minutes.
Incubation, dispense one microliter of reaction mixture onto a microscope slide and add one microliter of nuclear dice solution. Place a cover slip and check for nuclei formation via fluorescence microscope. Typically, round nuclei will form after 30 minutes of incubation indicating DNA replication has initiated.
Also, evaluate 10 microliters of the reaction mixture by immuno blotting using anti check one antibodies. Combine 50 microliters of LSE, one microliter of energy mixture and 1.6 microliters of toto mycin stock in a micro centrifuge tube. Then add 1.25 microliters of either two micrograms per microliter, a T 70 DNA duplex or water for the negative control.
Incubate the reactions at room temperature for 90 minutes and flick the reaction tubes every 10 minutes. Now, add 10 microliters of the reaction mixture into 90 microliters of sample buffer for western blood analysis of check one protein. The damaged sperm chromatin or DNA damage mimicking structure effectively trigger the a TR check one mediated DNA damage checkpoint in the xop egg extract system compared to the control MMS induces.
Check one phosphorylation at Syrian 3 44, which is an indicator of a TR kinase activation. Similarly, a T 70 as a DNA damage mimicking structure also triggers check one phosphorylation. Once mastered LSE preparation can be completed in around one hour.
Since the quality of extract diminishes after approximately four hours, speed and efficiency is essential. These methods can be coupled with other approaches to answer critical questions and DNA damage checkpoint signaling. For example, an immuno depletion can be performed to test if a target protein is required in the DNA damage checkpoint.
After watching this video, you should have a good understanding of how to study the D damage checkpoint using pus extract as model system. Thank you for watching and good luck with your experiment.
