PROTO31OL 330 RESTRI31TION DI29EST - PDF document

PROTOOL 3 RESTRITION DIEST TEAHER VERSION GENOME GENERATION TEACHING THE PROTOCOL 3 RESTRICTION DIGEST PROTOOL 3 RESTRITION DIEST TEAHER VERSION 2 STUDENT PRE-REQUISITES Pror to mplementng ths lab, students should understand • The central dogma of how DNA bases code for mRNA and then for protens • How DNA samples were collected and prepared forPR • The steps that occur durng the process of polymerase change reacton (PR) • What restrcton enzymes are and how they work • How the sequence varants n OXTR and YP219 are affected by restrcton enzyme dgeston • The purpose of PROTOOL 3 s to determne genotype STUDENT LEARNING GOALS 1 Perform restrcton dgeston of PR products of YP219 and/or OXTR 2 Descrbe the possble genotypes for &

#31;ndvduals wth the YP219 and/or OXTR genes 3 Predct what each genotype wll look lke after gel electrophoress and why PRE-REQUISITES & GOALS 3 Use the plannng notes space provded to reflect on how ths protocol wll be ntegrated nto your classroom You’ll fnd every course s dfferent, and you may need to make changes n your preparaton or set-up dependng on whch course you are teachng ourse name 1 What pror knowledge do the students need 2 How much tme wll ths lesson take 3 What materals do I need to prepare n advance 4 Wll the students work ndependently, n pars, or n small groups 5 What mght be challenge ponts for students durng ths lesson CURRICULUM INTEGRATION 4 REQUIRED LAB MATERIALS Ice bath or crushed ce Markers f

or labelng loves Amplfed DNA samples from PROTOOL 2 PROVIDED BY JAX For these mterls plese contct ttggxorg Mcroppettors & tps (1000, 200 & 20) 02 mL tubes n strps Tube holders/racks Restrcton enzymes (on ce) Thermal cycler Mn-mcrocentrfuge MATERIALS WORKSTATION NEEDS Dstrbute these mterls to ech workstton Mcroppettors and tps 02 mL tubes n strps Tube holders Markers for labelng rushed ce/ce bath Restrcton enzymes (on ce) Amplfed DNA samples PROTOCOL STRUCTURE STEPS 1-6 15 mnutes Break pont samples can be stored at 4º for up to 48 hours STEPS 7-12 2 mnutes to start, 40 mnute ncubaton perod Students do not need to be present 5 PROCEDURE The ability for a restriction enzyme to cut a PCR

product depends on whether the genetic variant creates or abolishes a restriction enzyme recognition site. PLANNING NOTES 6 STEP 1 Obtan a 02 mL tube strp and label them wth the amplfed DNA sample numbers STEP 2 Usng the P20 mcroppettor, transfer 10L of each of the amplfed DNA samples from PROTOOL 2 to the fresh tubes NOTE: Negative control samples are also processed through this procedure. STEP 3 Usng the P20 mcroppettor, add 1 L of restrcton enzyme to each new tube that contans PR product FOR YP219 SmaI FOR OXTR BamHI NOTE: Enzymes must be kept on ice at all times. WHY Restrcton enzymes, derved from bcter, hve dfferent DNA sequence recognton stes The enzymes below re specfc to the ste of the llelc vr

;nt beng studed n these genes STEP 4 heck that tubes are tghtly capped and gently flck the tube to mx STEP 5 Place tubes n the mn-mcrocentrfuge outftted wth the strp tube head Balance wth tubes on both sdes STEP 6 Spn the tubes brefly n the mn-mcrocentrfuge to collect the soluton n the bottom of the tubes NOTE If the end tab of the strp ht the top of the mn-mcrocentrfuge and prevent spnnng, they may need to be bent down orremoved PLANNING NOTES 7 BREAK POINT IF NEEDED The DNA sample can be stored for up to 48 hours at 4° C (refrgerator) Expected result is to have one tube per DNA sample (plus negative control) with 11 L of reaction solution (should be red in color). On the next page, choose the appropriate procedure for the gene of interest. WHY Restr

cton enzymes lso hve dfferent envronmentl condtons tht re optml for enzymtc ctvty You must choose the procedure pproprte for the enzyme you re workng wth FOR CYP2C19 (SmaI) STEP 7 heck that tubes are tghtly capped to avod evaporaton STEP 8 Incubate tubes at room temperature for 30 mnutes BREAK POINT The reacton wll proceed for 30 mnutes Samples can be stored for up to a week at 4° C (refrgerator) or can be stored at -20° C (freezer) for up to 5 years PLANNING NOTES 8 Expected result is to have one tube per DNA sample (plus negative control) with 11 L of reaction solution (should be red in color). Nothing should look different about the solution after the restriction digestion. The samples are now ready for PROTOCOL 4– GEL ELECTROPHORESIS FOR OXTR (BamHI) STEP 7 The thermal

cycler provded by JAX has been pre- programmed wth the restrcton dgeston protocol CUT  Dgests PCR products yclng condtons 1 Dgeston 2 Proten degradaton 3 Fnal hold forever STEP 8 usng PTC 1000 1 Turn on the thermal cycler usng the swtch n back and wat for the machne to run a self-test 2 heck that tubes are tghtly capped to avod evaporaton, place the tubes n the thermal cycler and close the ld Wth the cursor blnkng on RUN, ht PROCEED Select the approprate protocol for amplfcaton of your samples by usng the arrow keys and ht PROCEED  Prompt wll ask f you want to enable the heated ld, ht PROCEED  PLANNING NOTES 9 STEP 8 usng T-100 1 Turn on the thermal cycler usng the swtch n back

2 heck that tubes are tghtly capped to avod evaporaton, place the tubes n the thermal cycler and close the ld On the touch screen select Saved Protocols  Select the approprate protocol and press Run  STEP 8 usng mnPCR 1 Plug the mnPR nto the computer and turn on the thermal cycler usng the swtch n back 2 Open the mnPR software 3 If the approprate protocol does not exst, create a new protocol usng the PR template Input the name of the protocol, tmes and temperatures ndcated above for each step Save the new protocol 4 Double clck the approprate protocol 5 Select the mnPR(s) to run the program on and clck OK 6 heck that tubes are tghtly capped to avod evaporaton, place the tubes n the thermal cycler and close

the ld 7 After two mnutes of the program runnng, you can unplug the mnPR from the computer and t wll stll run the desred program, or f you keep t plugged n, you can watch the temperature cyclng on the software BREAK POINT The reacton wll proceed for 40 mnutes Once the protocol has completed, t wll hold a constant temperature of 4°  untl samples are removed (except the mnPR platform)  PLANNING NOTES 10 It s best to remove the samples and turn off the machne wthn 24 hours of completng the run to avod excessve condensaton accumulaton on the machne However, samples can be left over the weekend, f necessary STEP 9 Remove the samples after the protocol s complete, stop the program and turn the machne off Samples can be stored for up to a week at 4° C (ref

rgerator) or can be stored at -20° C (freezer) for up to 5 years Expected result is to have one tube per DNA sample (plus negative control) with 11 L of reaction solution (should be red in color). Nothing should look different about the solution after the restriction digestion. The samples are now ready for PROTOCOL 4– GEL ELECTROPHORESIS Sources of Potental Error The most common errors for PROTOOL 3 nclude • ncorrect mcroppettng • usng the wrong restrcton enzyme for the gene of nterest • not keepng the enzymes on ce whch reduces cuttng actvty Clean up Dscard all used tps and tubes n the trash except the DNA sample, amplfed reactons and dgested reactons PLANNING NOTES NEED HELP Emal the experts – ttggxorg TEACHING THE GENOME GENERATION | THE JACKSON LABORAT