hello uh today we are going to uh uh learn the experiment one with the title introduction tools used in experiments today's content will cover about the experiment objectives tools and their procedures and then experiment objectives the students are hopefully familiar and understand the basic principles of tools used in biochemistry experiments and the students are expected to use the tools properly during experiments we will cover four common tools used in pharmaceutical biochemistry labs such as micropipette ph meter centrifuge and sds page electroporator the first tool that i'm going to explain today is a micropipette micropipette is a tool used to transfer a small amount of liquid less than one milliliter with a micro liter scale usually in a lab there are three types of micro pipette with the p1000 scale and p200 scale and p20 scale as we can see here on the left picture the micropipette has six uh part the first one is a plunger at the top one uh the plunger is a button used to eject the liquid and this plunger is has uh important uh information such as we can see on the middle picture that the plunger has a word p1000 for example it tells you about the scale of the micropipette and the second part of the micropipette is a tip eject button this button is used to unplug the tip a tip at the bottom of the micropipette and then the third part is a volume adjustment dial this volume adjustment is used to increase or decrease the volume of liquid so you just can roll it up to minimum or to maximum one and the fourth part is about the volume readout the volume readout is used to see the maximum volume or the the number of volume that we can take it out from the liquid and then the next part is the tip eject shafts this is like a thin thin plate that help the tip eject button to eject the or to unplug the tip and the last part is at the bottom one is the tip attachment this part is a place for for us to put our tip on and moving on to the right picture there are three types of micropipette here so p1000 has the maximum volume is 1000 microliter or one milliliter and it has the accuracy ranges between 200 and 1000 and while the middle one the p200 it has a maximum volume is 200 microliter with the accuracy ranges between 20 microliter to 200 microliter and the last one is the p20 with the maximum volume is a 20 microliter and the accuracy ranges is between 0.5 microliter to 20 microliter as we can see uh in the middle uh volume rate out here for each of the micropipette there are three numbers there usually they have typically black black and red and the change of the volume adjustment is gonna change the number of the volume here like for example in b1000 the maximum volume at the top of the volume root out uh uh box here it's gonna tell you about one thousands microliter so if this show you 9 and 9 and 2 and 2 it means that there are 922 microliter but here as you can see that there is zero two and two so it means the first one is a thousand point and the second one is a hundred point and the last one is a ten point so it's same to the p2 200 and p20 so as a node it is prohibited to exceed the upper or the lower limit of your micropipette because if you exit the upper and lower limit you can damage the micropipette or microbiota will be totally broken and be careful be careful of a plunger stop point so actually the plunger has a two uh stop point there uh the first one is the first resistance point so it tells you how many micro liter you will go going to take out from the sample and the second one is the last stop point that you can barely push the plunger button so be careful for the first stop point and the second one and then next one is be on volume range don't go the lower limit or above the upper limit and for the next one is don't turn the pipette upside down because if you are working in a sterile condition that will contaminate your sample so the next part is if you're going to use the micropipette to taken out some liquid so first press the plunger until it reaches the initial resistance point so if you press for the first stopping point and dip the top of the tip into the liquid just barely below the surface now remember just barely below the surface of the liquid and avoid touching the bottom of the tip the bottom of the liquid container so uh when as you can see here on the right picture there is a step by step how to use the pipette uh correctly so the first is uh the first resist stop point and the second one you can just blow out all of the sample from the micropipette so if you are going to use the micropipette just press the first stop point and put your tip and dip your tip into the sample chamber and the step two is release the plunger slowly until it reach the maximal volume and the step 3 is taken out the tip slowly from the sample chamber and the step 4 put it or move the liquid you want to transfer into a new chamber and then press it slowly until it finish until the liquid is totally transfer into a new place and release for the step five release the plunger also slowly and then the step net the last step is just push the eject button to unplug the tip and moving on for the second tool is the ph matter the edge matter is a scientific instrument that measure the ph or acidity or alkalinity of the buffer solution these two as you can see here on the left picture it has two important part the first one is electrode part that connected to an electronic meter here that can show you that can display the ph reading here the part of the electrode one is sensitive to h ions and contains adcl and hcl if you are going to use the ph method there are two there are the first thing you have to [Music] remember that the ph meter have to be calibrated before you use it so the ph meter calibrated is using two or three different buffer solutions to reach the designated range for example you are going to check the sample between the ph 8 so you need three different buffer solution between the 4 and the 10 because your example is eight right and usually for a calibration they use the ranges ranges periods from four seven and ten sorry and usually if you are doing the experiment offline we need to do some experiment called ph determination so all you have to do is just uh determine all of these three four different buffer using ph meter and you can just write it down the ph method here on the column ph measure and moving on to this centrifuge uh centrifuge is a machine to separate particles within a solution due to the gravitational force and a centrifugal force depending on the particle size density and motor speed there are so many types of centrifuge we can find in anywhere in in the laboratory but basically they have two different types which is uh the gravitational force type and the centrifugal force type we will when we are talking about the rotor or motor inside the centrifuge so the centrifuge has two types of common motor centrifuge the first is a swing out motor in the right picture here and the second one is an angle fixed motor centrifuge on the left picture here so the difference between two is that the first the swing out motor it has the tube pocket that can that stick to the rotor that can move freely when the centrifuge motor rotates the tube inside the centrifuge will be in a horizontal orientation due to the centrifugal force and the second one is the fixed motor or angle fixed motor that enabled the tubes unable or cannot spin freely inside the machine and it's due to the gravitational force uh the advantage of using this type of centrifuge is that the speed of the motor is faster since there is a little two or no air friction in a centrifuge machine so if we are see if we see here uh before and after centrifugation so before the centrifugation process the cell homogeneity is spread all over the tubes but after the centrifugation there is two different uh part of the tubes so the palette then so this this uh these tubes contains two layers here the supernatant this is always uh in the first layer that because it uh contains a smaller and less tense component and then the palette one is the larger and more dense component so the next one we're gonna talk about the unit symbolize the force of the machine to separate the solution the centrifugal force or stated sg which is affected by the motor speed and radial distance of r of the particle from the rotation axis the g unit can be converted into rpm or rotation per minute and converted into rcf or relative centrifugal force according to its speed their centrifuge a different centrifuge as follows the first one is microcentrif centrifuge which has a maximum speed of 12 000 rpm that's why it's called microcentrifuge and the high and the second one is a high velocity centrifuge which has a maximum speed of further micro centrifuge one is a 25 000 rpm and a centrifuge force of 60 000 g and the third one is ultra centrifuge has a maximum speed of 18 000 rpm so the ultra centrifuge is way faster than the other two and when you are using the centrifuge machine there are several considerations upon to use that machine the first one is the centrifuge machine will operate when the lead is totally closed and the tubes are aligned and balanced so it is necessary to weight the tube before putting it into the machine to minimize turbulence to meet the balance requirement because unbalanced condition will enhance the turbulence and disrupt the sedimentation of the pellet that causes it to undo so before you use the machine it's better for you to weight your sample and make it balance between one and the second cell sample if you only have one sample just make sure to create the balance by using any liquid for example using water or something and this consideration number two it is better to fill up the centrifuge tube two or three quarter of the tubes to prevent bursts that cause a gastath so please don't fill up until full and the third consideration is make sure that the motor has completely stopped before opening the lid because this is totally dangerous and if you are actually if you are in a laboratory for the experiment you need to do this uh experiment on the left using two different samples here using the centrifuge upon these two different speed and you can tell the supernatant or palette observation like the the volume of the supernatant and how many palette you got so moving on to the last part is sds page electro operator so sds page or stand for the sodium dodecyl sulfate poly acrylic acryl acrylic limit gel electrophoresis is a technique of protein separation based on their molecular weight so sts ph is a technique of protein separation by their molecular weight this technique is widely used in forensic genetics by technology and molecular biology to separate the protein molecules based on their electrophoretic mobility the principle of sds page states that a chart molecule migrates to the electrode with the opposite sign when placed in electrical field so you can just imagine that the molecule is migrate and the smaller molecule migrate faster due to and the smaller molecules migrate faster due to less resistance during electrophoresis so we can see here the on the picture there are several parts of the sts page electroporator the first one is a casting stand here like a white plastic here the white transparent plastic here casting stand is you use just for the gel mold stand so you're gonna put some gel and stand standard gel on this casting stand the second part is the casting frame here and a plate glass like a sandwich so there is a glass a two to two type of glass that you stick it together and between the glass you can put the sds solution here and then the casting frame will press it and put it in the casting stand so we're going to explain it later and the second part the third part is a plastic comp here is a plastic to make a well before a sample so it's like yes it's like a com looks like it comes here and then the fourth part is a glass plate with integrated spacer here a glass to make an sds gel and the next one is a sample loading guide here it's a guide for loading the sample so you can just put your sample by using that sample loading guides and then the last one is a clamping frame here an electrode assembling here it's a box for the gel and its electrode after you set up with the plate you can put it here with the sample loading guide and put this one inside this chamber so i'm going to explain you a little bit detail here so the first before you are making the gel just clean the apparatus with the 70 ethanol in a picture and a b put the glass into the casting frame like this put the two uh different glass into this casting frame and the sea look the glass in the casting frame a lot i mean sorry lock the glass in the casting frame and put a casting frame in this casing stand there and do a leak test using water because if it's leaking then you cannot make any sds gel because the liquid will be run over the uh the glass the glass uh the glasses and then the next one is a take an acrylic mic solution using a micropipette drip the solution starting from the resulting gel the bottom one and up to the uh eight eight eight centimeter for example and until the stacking gel the upper upper part of the acrylamide solution or the sts gel and lastly place the con so the comb is the last part and wait for the gel to polymerize and the b picture the next picture is install the glass plate that has the gel to the clamping frame and electrode assemble and then the next is next step is pour the running buffer and get the com then clean off the well using the micropipette so it means that after the electrophoresis gel is tied up and put it in chamber and take out the cone and then clean the well using micropipette until no bubble and the last one pipette each sample one by one into the wall and it's totally done okay thank you for listening stay healthy and see you