Molecular biology Lab protocol

Molecular biology Lab protocol

• 1.
  My Mediums andcomponents YPD & YPDU Yeast extract : 1% Peptone : 2%. Glucose : 2% Agar : 1.75% For YPDU Add 2 μl of uridine per 1ml medium . from uridine stock YT & YTA Yeast extract : 1% Tryptone :0.5% Nacl :O.5%. Agar : 2% YTA : Add 1 μl of ampicillin for 1 ml of medium from the NOTE : Uridine can be added to the medium and autoclaved, ampiciilin should be added when media is at temp > 40 º in laminar air flow .ampicillin is heat sensitive and gets inactivated @ 45º. Uridine stock prepeartion : add 2.5 gms uridine to 100ml distilled water and autoclave 1. So 25 mg /ml & 25μg /μl( in media concentration should be 50μg /ml ) so add2 μl/ml of media Ampicillin stock preparation: 1 mg /10ml that is 100 μg / ml of media . so add 1μl /ml of media . 100ug/ μl For preparing the broths remove agar from the components and weigh them with the remaining .components . L.SORBOSE MEDIUM Yeastnitrogenbase (YNB): 0.17gms Ammonium sulphate :0.5 gms L sorbose : 2gms Agar : @2% Uridine :200μl Per 100 ml of distilled water Ura- YNB : 0.17gms Ammonium sulphate :0.5 gms URA- mix : 25mg Glucose :2gms Agar : @2% Uridine :200 μl Per 100 ml of distilled water
• 2.
  My Trp- YNB: 0.17gms Ammonium sulphate :0.5 gms TRP- mix : 25mg Glucose :2 gms Agar : @2% Uridine :200 μl Per 100 ml of distilled water COMPOSISTION OF H17 BASE : SC Sc mix :25mg YNB : 0.17gms Ammonium sulphate :0.5 gms Glucose :2 gms Agar : @2% Glucose :2 gms Uridine :200 μl Per 100 ml of distilled water SD MEDIUM YNB :0.17 gms Ammoniumsulphate :o.5 gms Glucose :2.0 gms Agar :2.0gms Uridine :200 μl NOTE: When agar is giving problem add low EEO agarose , (1.5%)for solidifying because , spatula contamination can happen or sometimes the agar itself may be contaminated in the manufacturing process and may give a back ground growth . So when there is problem with agar add agarose to nullify that effect. My protocols in my research 1) plasmid isolation: Inoculate the plasmid in to YTA and keep the flask on shaker at 37 deg for abt 5-6 hrs ( OD 0.4- 0.6) , aliquot as 1.4 ml in to ependorff tubes and collect the cells by centrifuging at 5000 rpm for 5 [email protected]º To the cell pellet add 100μl of soln I 200 μl of soln II( observe for the formation of clear solution )
• 3.
  My 1500 μlof soln III( observe for the formation of white ppt due to renaturation by potassium acetate ).  Vortex carefully on ice making sure that 4º temperature is not disturbed  Then centrifuge at 13,000rpm for 12 mins @ 4º and collect the supernatant in to fresh ependorff tubes .( make sure that the floating particles will not be pipetted out and the supernatant obtained should be 400 μl leaving the 50 μl in the tube )  Add ½ volume of tris saturated phenol and vortex it min 3 times on ice and keep the tube back on ice for 2 mins for resaturating the temperature.  centrifuge at 13,000rpm for 12 mins @ 4º and collect the supernatant in to fresh ependorff tubes.  Add 2.5 volumes ethanol (1ml) and keep in - 20 º for 1 hour (plasmid should be visible as a filamentous white ppt floating in the tube .  Again centrifuge at 13,000 rpm at room temperature for 10 mins and discard the soup , plasmid is observed as a white pellet sticking to wall of ependorff tube.  Dry the plasmid till the ethanol is completly dried and dissolve in 60 μl of TRIS –EDTA(TE ). Store the plasmid at -20 º till it is used up for the next step Solutions in this SOLUTION I : (TEG) : 50mm glucose , :25mm TRIS HCl. :10mm EDTA. MAKE 1 M stock solutions of this and dilute them as 5ml glu/ 100 ml , 2.5ml TRISHCL/100ml, EDTA 1ml/100ml SOLUTION II :10 % SDS, 2M NAOH = 10X SOLUTION II (dilute it 10 times before use ) SOLUTION III : 5M POTTASIUM ACETATE , ( 60 ml of potassium acetate + 11.5 ml conc glacial acetic acid + 28.5 ml sterile distilled water ) Note : solution 1&3 should be autoclaved and stored @ 4º, solutionII should be stored at room temperature and should not be autoclaved . Important observations and my own points Autoclave the glucose separately and add it in LAF, otherwise there will be charring.
• 4.
  My After solution2 clear solution should be formed , if the conc. of cells is more that is if the culture is over grown we still see some turbidity but if it is normal culture then clear solution will be formed . After adding solution 3 then there will be a formation of visible ppt , indicating that genomic dna is pptated and separated out . After adding 3 solutions and centrifuging , if there is formation of tiny particles again centrifuge the collected supernatant at 15,000. Make sure that phenol treatment , solution 2 addition are done to only cool tubes bz they can denature and break the plasmid . After adding ethanol and storing @ -20º t dna can be seen as white thread like ppt . and it itself validates that there is plasmid if we acn see that there is no need to run the gel fa checking the presence of plasmid . If phenol treatment is not done then if particles are there in ethanol do differential centrifugation to separate out them . Make sure that DNA will not exposed to too much time foer drying because the pellet becomes hard and will be difficult to dissolve .and there will be much chances for tip contamination. Always keep the ependorff tube in such a way that the opening should be facing our side so that the plasmid can be located easily( pellet will formed on the opposite side of the opening exactly at bottom ) , this technique helps us to dissolve the plasmid properly particularly when the cell concentrations are low and when very low amounts of plasmid is formed . Dissolve the plasmid in 6o μl of TE , min 50 μg of plasmid should be there to be visible in the gel . so don’t dilute it too much . if for dissolving, when plasmid is too much diluted use ethanol to repreciptate it and concentrate it. After adding solution III give it a 10 minutes incubation in ice , this will precipitate the proteins , DNA , high molecular weight RNAS . SOLUTIONS AND BUFFERS NEEDED FOR PLASMID PREPEARTIONS Solution 1: per 500 ml: 50 mM glucose 9 ml 50% glucose 25 mM Tris-HCl pH 8.0 12.5 ml 1 M Tris-HCl pH 8.0 10 mM EDTA pH 8.0 10 ml 0.5 M EDTA pH 8.0 Solution 2: per 500 ml:
• 5.
  My 1% SDS 50 ml 10% SDS 0.2 N NaOH 100 ml 1 N NaOH Solution 3: per 500 ml: 3 M K+ 300 ml 5 M Potassium Acetate 5 M Acetate 57.5 ml glacial acetic acid TE per 100 ml: 10 mM Tris-HCl pH 8.0 1 ml 1 M Tris-HCl pH 8.0 1 mM EDTA 0.5 ml 0.5 M EDTA pH 8.0 Candida transformation:  Inoculate the strain (CAF4.2) and allow it to grow @ 37 º for 6-8 hours, collect the cells by centrifuging at 5000 rpm for 5 mins .  Add 1 ml of 0.1 M liac and leave for 5 mins and wash the cells properly in 0.1 M liac to remove the traces of media .  Collect the cells by centrifuging at 5000 rpm for 5 minutes at RT , and resuspend them in 200 μl 0.1 M liac and allow them to be saturated for 2 minutes .  Aliquot them as 100 μl in to separate ependorff tubes and add the following components . 
• 6.
  My POSTIVE CONTROLnegative control 240 μl PEG 240 μl PEG 34 μl liac 34 μl liac50 μl plasmid (PRC 2312, PRC 3915, PCA 88) ----------- 36 μl autoclaved water 86 μl autoclaved water  Vortex it for 3 times min. To mix the components properly make sure that PEG and LIAC gets mixed properly so that LIAC can reach the cells to make them competent  Leave the tubes from 1 hour to over night incubation at room temperature .Heat shock is given at 44 º for 25 minutes or at 42 º for 40 minutes .  Wash the tubes properly with water and centrifuge at 5000 RPM to collect the cells , and then redissolve in 50-70 μl water basing on cell concentration and plate them on URA- plate for selection for 72 hours and see for the result . My points :  The efficiency will mainly depend on the pre incubation, because the PEG added is viscous and can hinder the 1M LIAC from reaching the cells . so the pre incubation remains the key for the transformation efficiency .  All the steps should be done at room temperature.  OBSERAVTION AND RESULT: On positive control, there should be transformed colonies and in negative control, there should be no colonies. MAKING REAGENTS : PEG 800: add 50gms to 30ml glass sterile water and boil it till PEG is completely mixed in water and add 70 ml of autoclaved glass sterile water .( TEG can be a source of contamination so as soon as u do it , aliquot it in to epenodrffs and transfer to -20 º & make sure that it comes to room temperature before use , as we use single tube 2 times there will be less chances of contamination ). LIAC : add 102 gms of liac in i lit distilled water and autoclave , store at 4 º , this is 1M solution , dilute it 10 times with sterile glass distilled water to make it 0.1M .
• 7.
  My E.COLI TRANSFORMATIONμl, º  Inoculate the appropriate strain (XL1, JM101, DH5α) from on culture in to fresh 25 ml YT liquid ( add ampicillin @ 1 μl/ml) and allow them to stay at 37 º for 4-6hours till OD 0.4-0.6.  Transfer the tube in to an ice box and pre cool it for 1 hr till all the cells gets equilibrated to 4 º.  Transfer the culture in to autoclaved 50 ml centrifuge tube and centrifuge @ 4 º for 5 minutes to collect the cells.  Immediately keep the tube back in ice and transfer the ice box in to LAF and then add 800 μl of 0.1 M cacl2 solution to it, suspend the cells in calc2 and leave it on ice for 5minutes for resaturation .  Aliquot this in to ependorff tubes 100 μl each and add 10 μl plasmid DNA to positive control tube and add 10 μl water to negative control tube.  Incubate on ice for 30 minutes, and give heat shock by keeping it exactly at 42 º. For 90 seconds.  Keep the tube in ice for 2 minutes ( cold shock ) and transfer to LAF . to this add 4 times 400 μl YT and incubate @ 37 º for 45 minutes .  Plate 100 μl from this and plate on YTA plate. Observation and result :  Colonies should be formed in positive control and no colonies should be there in negative control. My points :  Never cross OD 0.6 when preparing the cells , because after this valve cells will pass the late log phase and will not give good efficiencies during transformations . sometimes they may not give any transforments too .  Everything should be properly autoclaved and pre cooled that was the key . every component and tube or tip used for this procedure should be @4 º .  Ensure that heat shock is appropriate. Moreover, the cold shock should follow the heat shock.
• 8.
  My  Transferredplasmids should be selected basing on markers and making the media with drug that can select recombinants.  Never forget to add the antibiotic prior to cell inoculation in to flask .  Plasmid should be of good quality and cells should be late log phase cells ( late log phase is achieved for XL1 after 4 – 5 hrs incubation @ 37 º)and when plasmid is bound to proteins in its preparation , they may be hindered at cell walls or may not be expressed in host cells due to the attached proteins) .  When selecting for ampiciilin make sure that the ampiciilin is above 100μg because the tarnsformants may use up the ampicillin and may give satellite colonies in their vicinity by forming media zones without ampiciilin around them.  If this happens a false transformant can be selected for a true transformant for further analysis . Making agarose gels for gel electrophoresis: O.5 X TBE IS the working buffer to which 0.8%- 1.2% agarose high EEO(electro endo osmotic is added ). TBE ( TRIS- BORATE – EDTA ) is the standard buffer used with the following composition. TBE BUFFER COMPOSISTION Tris :54 gms Boric acid :27.5 gms EDTA : 20ml( 0.5 M ) Make the volume to 1 LITER DISTILLED WATER . Procedure : BPB :4mg/ml dis water ETBR :10mg/ml in distilled water  Take a clean measuring cylinder and rinse it with distilled water 2 times , and measure 36ml of sterile glass distilled water and pour it in to pre cleaned beaker ., to this add 4 ml of 5x TBE .( the resulting will be working solution of TBE)
• 9.
  My  Weigh0.32 gms of agarose and add it to working solution , melt the gel in microwave oven and add 15 μl ETBR .  Pour it in to a gel casting tray and arrange the combs properly , allow the gel to solidify .  Take 10 μl of DNA sample and to this add 5 μl of distilled water and 3 μl of 6x BPB( bromo phenol blue .  Load approximately 15-25μl DNA in to wells and run it at 110 volts till the bromophenol blue reaches 1/3 of the length of the gel .  Carefully remove the gel and see it on an UV TRANSILLUMANTOR under a black back ground . My points : 50 nanograms of the plasmid should be loaded at least to see the bands on transillumnator .  Load a marker always along with DNA samples to validate the correct plasmid by band intensities and molecular size .  10 kb ladder shows the band where plasmid is separated and it also shows the gel is correct when the ladder is separated.  Loading marker : To the supplied marker add 3 μl of BPB and 5 μl of distilled water and load it along with sample for authenticating the sample . NOTE :  make sure that the casted gel is kept from – to + end always ( because DNA always run from negative to positive )  make sure that the cellophane tape used for casting is removed before placing the casting tray in to electrophorotic apparatus .  While loading never suck the buffer in to the tip , if buffer is sucked the sample will spill from the wells . RESTRICTION DIGESTION : Transfer the following components accordingly in to an 1.5 ml ependorff tube . Plasmid DNA = 10μl RNASE =2 μl RESTRICTION ENZYME =1 μl
• 10.
  My BUFFER =2μl WATER =5μl TOTAL VOLUME =20 μl Keep for incubation from 1- 4hours depending on the concentration of DNA being used and depending on the enzyme purity . Note : when checking for cross contaminations keep the sample for overnight incubations with restriction enzymes .so that the effect of the second enzyme which is present in trace amounts will become evident . so that 2 different bands should be formed in gel . it tells that the restriction enzyme is contaminated with another enzyme due to tip contamination or due to a contamination in the process it self . Theory :  Plasmids are restriction digested to validate the quality of plasmid and also to check whether the isolated plasmid is correct or not . correct plasmids should release the desired fragment when digested with appropriate restriction enzymes . this is how the isolated plasmids are validated .  If the isolated plasmid is bad in quality restriction digestion will be hindered and there will be no result with the restriction enzymes when digested .  Buffer combinations should be choosen correctly, see for the colour codes always TNAGO buffer (fermentas) is feasible with 98% of the restriction enzymes but gives a different percentage of activity with some of the enzymes in that case the initial concentration of the enzyme should be increased to avoid the problem.  In double digestions see for the enzymes that work with a common buffer ,this is very important& if the combination is not right one of the enzymes will work and the plasmid will be nicked , but no fragment will be released . ADDADDARERESTRIC
• 11.
  My Haemocytometer PRINCIPLE The ruled area of the hemocytometer consists of several, large, 1 x 1 These are subdivided in 3 ways; 0.25 x 0.25 and 0.20 x 0.20 mm (0.04 mm further subdivided into 0.05 x 0.05 hemocytometer hold the coverslip 0.1 defined volume. Dimensions Area Volume at 0.1 1 x 1 mm 1 mm2 100 0.25 x 0.25 mm 0.0625 mm2 6.25 nl 0.25 x 0.20 mm 0.05 mm2 5 0.20 x 0.20 mm 0.04 mm2 4 0.05 x 0.05 mm 0.0025 mm2 0.25 The cell-sized structures counted lie between the middle of the three lines on the top and right of the square and the inner of the three lines on the bottom and left of the square. In an improved Neubauer hemocytometer (common medium), the total number of cells per ml can be discovered by simply multiplying the total number of cells found in the hemocytometer grid (area equal to the PROCEDURE mm (1 mm (0.0625 mm2), 0.25 x 0.20 mm2). The central, 0.20 x 0.20 mm marked, 1 x 1 mm (0.0025 mm2) squares. The raised edges of the mm off the marked grid. This gives each square a mm depth nl nl nl nl cytometer red square in picture on right) by 10 mm2) squares. ), mm (0.05 mm2) mm square is ) 104 (10000).
• 12.
  My Ensure thatthe special coverslip provided with the counting chamber (thicker than standard coverslips and with a certified flattness) is propperly positioned on the surface of the counting chamber. When the two glass surfaces are in propper contact Newton's rings can be observed. If so, the cell suspension is applied to the edge of the coverslip to be sucked into the void by capillary action which completely fills the chamber with the sample. Looking at the chamber through a microscope, the number of cells in the chamber can be determined by counting. Different kinds of cells can be counted separately as long as they are visually distinguishable. The number of cells in the chamber is used to calculate the concentration or density of the cells in the mixture the sample comes from. It is the number of cells in the chamber divided by the chamber's volume (the chamber's volume is known from the start), taking account of any dilutions and counting shortcuts. In the most common design, the volume of each large square is 0.1 mm3. The cells in four large squares are counted and cells over or touching the lines on top and on the left are counted, but cells over or touching the right or bottom lines are ignored. The concentration in cells per ml = cells in four squares/4 × 10,000[1]. Easisest way will be mixing cells in to 1000 μl and taking 10 μl to count .so multiply the total by 106. Count 4 chambers and make the count as Q1 +Q2+Q3+Q4 106. 4 From this make serial dilutions so that 500 cells per plate can be plated. PCR : Procedure : Pre program the pcr by the given method : 1) Lid Pre heating 105 º 2)Denaturation 94 º
• 13.
  My 3)Primer aneling45-50 º 4)Primer extension 72 º 5) no of cycles 35 Components in to pcr tube . Water : 16.5 μl Buffer :2.5 μl Taq dna polymerase : 1 μl Primer R :2 μl (10 pico grams ) Primer R :2 μl (10 pico grams ) dNTPS :2 μl. Template :1 μl. Mgcl2 :1.5 μl Total volume : 28.5 μl  When designing the protocol normally extension step will be based on the amount of DNA getting amplified and the procesisivity of the TAQ DNA polymerase . normal valve is i1kb /sec. so basing on the kbs of region getting amplified the time limit will be fixed .  While designing the annealing time for primers always see for the TM of the duplex being formed and the valve should be less then that . if aneling temperature is over the TM , the primer /template duplex will not be formed .  Make a list of all the components being added and tick every component as soon as u add it the ependorff.  The cycle no will depend on the requirement of the quantity of the pcr product .normally for 1M concentration of the dNTPS 40 cycels is the higher limit after that the shorter fragments will start accumulating making the PCR product to be heterogeneous . so 40 cycles is the recommended no
• 14.
  My  Mostof the time genomic DNA is the culprit so make sure that the genomic DNA is of good quality with out proteins and RNA contamination .use RNASE , heat treatment for 20 mins 80 º , or use 3M potassium acetate to remove the proteins when ever required .