Molecular Biology Labs PDF

*. enters formentert Measuring weigh preparingaltions : ⑧.. Measuring weights : · Small units can be measured using high accurancy balances. e -...

*. enters formentert Measuring weigh preparingaltions : ⑧.. Measuring weights : · Small units can be measured using high accurancy balances. e - g : mg unit. - - 0.000 L 0.00000 The last digit is uncertain More digits = More accurancy · To measure tiny amounts Prepare a stock solution (substance distilled water) + with known concentration and volume , then calculate the mass using the formula : - Level indecator make sure that the bubble is in the middle of the circle -. 11111111111111111 Adjust the bubble's position by the legs of the balance. Heavy at ? back right capacity heavy at ↳ front left Must be in the middle Before using for accurate glance check I ⑳a readings : " 8@. I Level indecatOr , 1 Temperature , , , , · always cool the samples into room temperature · Some substances are easy to evaporate must being fast while weighing RANA s ⑤)|⑧ while ⑦@-⑦ dealing with balances : ⑧.* * * Always place the container in the middle of the balance. ⑧↳ Try to stick with the same balance for all of your readings ⑧ To check the balance quality weigh something , with known mass. ⑱ Preparation of solution : · Make sure to lable every solution with I main informations : name concentration substance name date accurately las!? metric measuring volumes The meniscus must be lying - !! down in the calibration mark. Rinse the into the volumetric flask · stirring rod to avoid losing any drop of the solution which might cause errors stirring rod distilled I water funnel volumetric flask RANA Measuring Mymes ⑬ : Measuring Volumes : ⑮ Large Volumes ↳ > ③ 01-25mL ↳ > - Graduated volumetric volumetric pipette cylinder flask (10mL-2L) (high accuracy) ⑬ Microliter range Never use These for ↳ > measuring volumes : - Beaker flasks The measures are aproximate Micropipette RANA  Micropipette Components Plunger button shaft Friction ring -Il. Tip ejector - Digital volume indecator Pipette tip ·ters Somemicropipetes have filters. to prevent any contaminatithe Micropipette Types 10 20-100ML -00 100-1000ML 40 : : 7 8-10.. 0ML : gri - I I - - I T O 5 O j1 - O 0 -15 - 0 fractional - > I 0 11111 1111111 11 K 111 1111111 I 1111111 part 11 III fractional K > part · If we want 20ML which one is better to use : 1) p20 (0. 2-20) - more accurate because it is the maximum reading 13) PIOU (20-100) more accurate :8 & S & S; 11 vi. " maximums 108 $ : S C) P200(20-200) RANA  How to use the micropipette : D Make sure of the & pipette's range. ⑫ AHatch the tip To prevent the substance from soft press reaching the inner ⑬ Press the on the plunger parts of the pipette and ⑲ Dip the tip in the liquid and release the plunger slowleyruinlyn the desired place and press the soft press ⑰ Move the substance to plunger with the hard press ⑱ Press to avoid losing any drop ⑳ After finishing , adjust the volume to the maximum value to avoid running the spring Dinner lays down every · the balancing drop is at very the bottom important of the tube (no vibration). - 1↓ - - C- d S i 11 I 3 - - I11... 0 o E S RANA  extraction LDNA : 'sprotection mechanism = ° @ ° @ =x= $ @ ° rokaryote a contains ristriction Doesn't have nucleus , = - Methyl group protect nuclear membrane enzymes degrade the cell from its own : , , any foreign DNA defenses Eukaryotes a · Surrounded by membrane bound nucleus. · plants have extra protection provided by the cell wall DNAse enzyme Must be denatured ↳ ⑱ - Miner - cytoplasm in ,* before reaching - W ↳ t- - ⑰Pe DNA... - * - ISell lysis · Bead beating the sample · Vortex with phenol (break call wall) · Detergent such as SDS (remove lipid membrane) risionof ⑰* ⑮⑦°π⑦ protein · Addion of salt JNAG , NaCICOO-I remove remaing protein) · Vortex with phenol-chloroform E centrifuged the protein (separate other nucleic acid from substances) RANA recipitation · Cold ethanol or isopropanol (increase the yuld of DNA -> DNA won't dissolve in ethanol) Washing · Cold alcohol (prevent denaturation of DNA) DNA re-suspension ⑮ Tris TE · or or ddHG DNA extraction - Phenol Chloroform method $DNAS18- ~ - !I DNAS125 : - s & Add the sample in Eppendorf tube * NapOn releaS e extracellular DNA : & Add NaPO buffer E TNS TNS : procedure of the * of DNA co-extraction ③ Use freeze and throw method to break cell wall xuse water bath liquid nitrogen not sold ④ Add proteinase E incubate x break down protein 50 Vortex 10 min ⑧ centrifuge > , · Umin , 13000 RPM Lic ,. ⑧ ⑧ ↳ ⑧ & Take amount of supernatant and place it in new eppendrof tube ⑳ Add phenol/chloroform/isoamy alcoholx separate nucleic acid from other substances Vortex ⑧Centrifugeumin a , 1500 M, ic · ↳ ↓I nier? a secre · -organic phase RANA  ④ Take amount of supernatant and place it in new eppendrof tube ⑫ Add phenol/isoamy alcoholx promotes the partitioning into organic phase b aqueous phase Vortex ⑧ centrifuge aqueous phase- / - & I organic 1 in UmIn , 13000 RPM UC ↑ , · %I an eraseSee · ⑧ ⑧ Take amount of supernatant and place it in new eppendrof tube ⑬ Add PEG x increase the overall yield (to precipitate DNA) Vortex ⑧Centrifuge zomin , 15000 PM , sc · ↳ -I ↳ ↓I ⑧ ⑧ Remove the liquid by pipetting and tissue 8Centrifugeumin Add cold 70 % ethanolx remove the shell arround DNA solvation ~Vortex ic B , 15000 PM, · I ⑧ Pellet ⑧Dry Add TE bufferx solubalize the nucleic acid while protecting them from degration (elute) ④ Store at -20° ⑮ RANA  mid ⑯5 : Genetic Component in bacteria -...- - =... Bacterial Extrachromosomal - chromosome · , is Plasmid a ! found in any extra molecule of DNA that bacterial cell enhance the cell chance of surviving : a resistance against antibiotics -si-- degrade invading genetic · - material a Bacterial chromosoma S ⑧ ⑧g n Plasmid >29 - ⑧ ey / - P of " replicate Is specific genes (proteins) that · amich a mportance =@ ° ⑦ ? - -"We isolates * less complicated - small circular DNA # · Discard supernatant from pellet cells · Resuspend cells in resuspension solution pipette up & down , or vortex Transfer centrifug homogenize * · to oak ridge style the solution Add lysis · solution & invert gently x break down the cell membrane E cell wall I contain SDS -min s & j · Add neutralization , invert multiple times ⑧ * change the pH to break down the bacterial chromosome RANA ioismin As 8 · · Transfare cover the cleared tube with lysate parafilm into midi spin column * to in prevent contamination collection tube. min As -j ⑧ · · · Add cold ethanol Add wash solution Discard the liquid E to x wash solution proteins removing add wash solution again smin s & Transfer to new tube & · Add elution solution * provide more purification. RANA  cation DNAqui : kantations 1) wear gloves 2) Do not cross-contaminate (for each solution use one pipette's tip) 3) Make sure that appendrof tube's hinge is pointing upward in centrifuge 4) Start by warming the spectrophotometer around 15 minutes with no curette inside ↓Bearing D sample · 10 ML of DNA with 990ML of distilled water - 1 : 100 dilution Hetometer · Absorbance of 1 relates to 50Mg/mL x 10D -> 50mg/mL eading sample ). > stand · Azgo : wavelength or concentration in OD · Azso : absorbance (inverse relationship with purity) · purity = Acco Az : the ratio must be 1 5 (best result. 1 8-2.. 0) Conc(mg/mL) = A2600D) + since charged DNA , E place them RNA are negatively cathode (-Ve) 1 , on. In case DNA or RNA were placed in anode +Ve) they would go , outside gel's frame seaavana I e disadvantages e a / 1 , 15115 , s , 11 111 / · Poured easily / · Melts in high temperature / / / · Has no affect on DNA / · Buffer needs to replaced / pe / / · samples can be easily / · Different genetic material / recovered / unpredictable forms. / / has / / 111111111111 , , , ,... , , , ,... 111111111111 Applications of Agarose Gel Electrophoresis B& Estimation of the size of DNA L 43 6 Analysis of PCR product A - of restricted genomic DNA ↳ separation RANA factors that affect DNA migration * engtDNA : x Short DNA-> moves faster x DNA- moves slower Longer & a concentration : check if DNA from - Note : to we have brittle -- concentration -> small molecules extraction use low concentration x High / , because it is molecule large of / * low concentration -> large molecules / agarose weak / -1111111111111111 ⑮ Hage : * High voltage - faster movement x High temperature -> Agarose melts x High voltage -> decrease the resolution. i Y oof n DNA plasmid : 0 t one heavy block Nicked Linear to long that could be disructed supercoiled same as yarn ball Visualization lib ur Ethidium Bromide CEBr) with density of 0 2-0 5 Mg/mL At inserted between under... the base pairs Under visible : ⑤je) Loading buffers(Xylene cyanol Bromopheno Blue) > B co-sediment with DNA-moves as the same speed of DNA RANA  myfer 11 111 '1 , by compenation 1 , of weak acid/base E conjugated base/acid that control pH. - E E E if the glycerol primary ~ is ; ⑮Bo - answBora , component of · lowest capacity · higher capacity , arge fragment e agarose · low voltage longtime , oborate reacts with (more than 5kbp) · best resolution for large sugar from the backbone o low conductivity and high molecules of DNA voltage · short time for routine work. ⑫ ⑤ ⑧fined & O↳ o ⑭ g ave - buffer I Ag arose.. wave # ize ⑮ ⑯ - ↓ ↓ ↑ , casting tray pouring agarose Gel combs make wells G ⑥ / > ⑧ ⑨ S..⑳ * - -⑭ 8 Anoce place the DNA at will amodrom RANA ↳ chain reaction Polym ⑬? Amplifying a peice of DNA in Cabs by enzymatic replication. mal cycling A machine which cool heat the PCR samples e " M ingrediys impot : - - - B DNA template ] aq polymerase & ~ III" i ~ high temperatures ] two DNA primers & " Howards backward) & dNTPs -sure ⑳ ilization & Heating reaction (94-96°) for[3-9 min) - I - ⑫ aguration & first step of cycle separation of, the #(1) ↳ two strands of DNA at (94-98°C) #(1) for E 20-30 sec - 1111111111 4 ③ Jing Annea Second step of cycle where the primers , - 11I #(1) take place on their complementary sequence at (50 56°) - for(20-40se() #11 I ⑭ Elongation Last step of cycle where the tag polymerase &I111 start the DNA synthesis by adding a proper I dNTP in each nucleotide at (75=80°) optimum A111I (72)for tag ⑤ inelongi To make sure that all single DNA strands are extended at (75-80°C) for 15-15min) ⑯ Singe hold storage for the samples at (M-15) at a time RANA  - " ~ xcycles duration nation final Elong ling a " a 00 : 30 C & Cagulating al time : single C cycle number of eces, holding e , "i RANA : DNA sequ eying : stermination Sanger sequencing - / / B A method to determine the precise order - method) of nucleotide fragments of DNA 1999 199999911" I Aerophoresis i -19933915 , y &&& CH NB &&& CH O NB &&& CH NB , O , , O A * * * * * H H H A H A O OH OH H-bind in ' H H bind in 3' E2 ribonucleoside deoxyribonucleoside dideoxyribonucleoside triphosphate triphosphate triphosphate (NTP) (dNTP) high concentration S (ddNTP) will the process stop from the beginning cuse low concentration) we only primer in sequencing to avoid doubling the bases · use one nitrogenous. · in 5.... CTGACTTCGA --- 3' & DNA sequence 3..... GDAPTGDAAGDPT1........ entery TAG C jj we - - - - ⑳ ⑳. ⑳ &. - - shortest - 5' 5 entery. 3.... GICTGAGCT.... -> N sequence RANA acrylamide can differ each fragment with only one nucleotide e Ited Sange , her See · Single tube with all daNTP , each type has unique fluorescent label. ddGTP · ddTTP 8 sample ddCTP · ddATD ↓ capa ? ↳ W ] or & - "o RANA