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=Image and Seq= *[[Matt:LabNotes/2017-6-8|Last time]] confirmed no polonies **Ideas for why: ***Gels were cast and stored poorly in water (UV slides beforehand, use immediately or store in buffer with EDTA) ***Primers on beads outcompeted primers in gel (USER digest amplicons on beads to shorten primer on beads) http://arep.med.harvard.edu/polony/polony_protocols/pcr.htm http://arep.med.harvard.edu/polony/polony_protocols/sbe.htm ==Sample Conditions== *3 samples to test how USER can help create polonies **USER will nick the biotinylated strand leaving only: CAGTGT Samples: #Basic repeat of last time with fresh gels #USER digest beads before adding to gel #Include USER enzyme in the PCR mix ==Cast 10% gels with primer== #Make Gel Mix #*25ul 199:1 40% A:B mix #*2ul 10% BSA #*67ul H2O #*2ul 50uM Acrydite primer #*2ul 5% TEMED #*2ul 5% APS #Add 18ul to oval and cover with coverslip #Put slides in argon chamber and polymerize 30min #Wash in H2O 30min shaker ==Prepare Beads== #Beads prepared on [[Matt:LabNotes/2017-5-24]] ===For USER prenicked Beads (Sample 2)=== *Take 5ul beads (1:1 mix of v1 and v3) *Add 2.5ul 10X Cutsmart Buffer *Add 2.5ul USER *Add 15ul H2O *Incubate 15min @ 37C *Put on cold block 5min *Pull down beads, remove supernatant, and resuspend in 5ul Dynabeads buffer **Was not able to pull down beads so just pipetted the reaction mix (1/5 diluted beads) ==Gel PCR== #Let gel slide dry in AirClean hood for 30min within 5 minutes of liquid film disappearance #Prepare 25ul PCR mix per gel #*2.5ul 5uM Primer2 #*12.5ul KAPA SYBR FAST MM #*5ul beads (1:1 mix of v1 and v3) #*5ul H2O #*For USER sample 3 include 1.25ul USER and use 3.75ul H2O instead #Pipet 25ul onto center of gel #Apply 18x30mm cover slip #Apply an orange SecureSeal chamber #Fill chamber with mineral oil and seal holes with stickies. #Slide PCR on Biorad thermocycler #*Put in sample 3 first, put in sample 1 & 2 after 37C 30min incubation 37C 15min -> 94C 3min -> (94C 1min30sec -> 55C 1min -> 72C 2min) x 20 -> 72C 4min -> 4C hold *Made a mistake and did 15sec 37C at first, didn't catch the mistake until after 94C 3min incubation probably killing the USER enzyme **Restarted the thermalcycle program anyway just in case but probably won't work #Place directly on Olympus with GFP/FITC filter ==Results== ===Sample 1: Standard=== *Like seen before, amplification on beads only [[File:20160613_Sample1_Standard_composite.jpg|450px]] ===Samples 2: Prenick with USER=== *Signal only in beads [[File:20160613_Sample2_PreUSER2_composite.jpg|450px]][[File:20160613_Sample2_PreUSER5_composite.jpg|450px]] ===Sample 3: USER in PCR=== *Again signal only in beads *Expected because not enough time for USER digest [[File:20160613_Sample3_USER_composite.jpg|450px]][[File:20160613_Sample3_USER1_composite.jpg|450px]][[450px]] ==Conclusion== *Need to try USER in PCR again *Try adding 1/10th free floating acrydite primer *Try following Harvard protocol more closely *Fresh gels made no difference
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