Outline for today. Review of events for this week Background material so you can understand DNA extrac=on and PCR

Transcription

1 Outline for today Review of events for this week Background material so you can understand DNA extrac=on and PCR

2 This week Monday (part 2/b): Examine extracted DNA by gel and spectrophotometer; set up PCR Weds (Ex. 10 part 3/c: day 1): Examine PCR product by gel and spectrophotometer; set up restric=on digest Next Monday (Ex 10 part 3/c, day 2: Run restric=on digest on gel, clean up PCR product and send out for sequencing

3 This week we con=nue Molecular Strain Iden=fica=on Goal is to use 16S rrna gene analysis to determine the iden=ty of your unknown strain. How is this done? Extract genomic DNA from pure single colonies Monday: Verify that you have DNA using (1) Gel Electrophoresis and (2) Spectrophotomer Set up PCR reac=on to generate PCR of 16S Weds: Verify that you got PCR products using gels and spectrophotomer Set up restric=on digests Send of for sequencing (next week); compare sequence to all others in the world (later this month)

4 Today: Check to be sure you got nice sized Genomic DNA Run on gel Compare to marker Some modifica=ons to lab manual 50 ml agarose instead of 75 ml Using Gel Red instead of Ethidium Bromide (2.5 µl) Image from:

5 Loading and Running Gels negative Samples go in wells Bigger Pipette in with 6X tracking dye, (purple color) Smaller positive =1&videos=V0VVjR0mv7c

6 Quan=fy your genomic DNA UV Spectrophotomer DNA/RNA absorbs strongest at 260 nm 50 µg/ml has OD of 1 at 260 nm. You can use this rela=onship to calculate DNA concentra=ons of an unknown samples. Pure DNA has a 260/280 of 1.8 Abs wavelength

7 Schedule today to accommodate plate pouring 1. Mix agarose and pour your gel needs ~20 minutes to solidify. 1 gel/group of 4. i. While that is cooling or before the class starts weigh out your media ingredients, assemble in flask ii. iii. Place on cart for autoclave Start autoclave 2. Load and run your gel, 60 minutes (autoclave going) 3. Nanodrop reading of your DNA (autoclave going) iv. Remove media from autoclave, set in baths to cool 4. Set up PCR aier you see that you have DNA (2/group) v. Pour your plates

8 Aier you know you have genomic DNA, the next step is to set up your PCR Ideally we will set up two PCR reac=ons/group, but if you only have one genomic DNA/group, just set up one.

9 Polymerase Chain Reac=on Amplifies DNA by making many copies Used for detec=on, engineering pieces of DNA, amplifying enough DNA to sequence it Many more applica=ons.

10 PCR: Exponen=al Amplifica=on

11 PCR: Ingredients DNA template. Primers: targets the amplified sequence Heat stable polymerase (Taq) Mg 2+ (cofactor for polymerase) Buffer (maintains correct ph) dntp s (datp, dttp, dctp, dgtp) Thermocycler: precise temperature control

12 Senng up the PCR!"#$"%&%'( )"*+#&(,-%.*(!"%/&%'0.'-"%( 12(%3456(7&#$*.'&(89:(( ;( 56( ( 12<(=&./'-"%(>+??&0( ;( 56( ;( 56( ;( 56( (,"0D.0B(C0-#&0(@(5A(EF?G( ;( 56( ( =&H&0I&(C0-#&0(@(5A(E1JK@0G( ;( 56( ( 9+/*&.I&(,0&&(L.'&0( 1KMN( 56( O( 7"'.*( JKMN( 56( ( E:BB&B(PQ(-%I'0+/'"0G( ( ( 7.R(89:(C"*Q#&0.I&(;S456( 2MT56( (,-%.*(H"*+#&( ;2M256( (

13 PCR: Thermocycle Thermocycle Profile 95 C Repeat 30X C Denaturing Annealing Extension Temp. 52 C Time hop://

14 !"#$ %#&$#'(")'#* %+&#*,)&-#'*./*0102#3* 4+5* 6#7(")'("+.7* 89:;* 9* &+7* <* 6#7(")'("+.7 8=:;* >?* 3#0* 9A:;* >?* 3#0* * BC"#73+.7 DA:;* A* &+7* * %E.'.)5E* BC"#73+.7 DA:;* 9* &+7* <*!".'(5# =:;* F.2G* *

15 PCR and Gel Electrophoresis

16 The next step is to check whether you got the right size of PCR Product using Agarose Gel Electrophoresis Agarose comes from seaweed and is similar to agar Agarose is added to a buffered solu=on in the range of 0.5 2% Genomic DNA: 0.7% 1 kb: % ******* What we will use for PCR product****** 100 bp: 2% DNA is stained with gel red (change in protocol) Needed for visualizing by UV on a transilluminator

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18 Aier you have PCR product, we will aoempt to see the class diversity using restric=on diges=ons Take PCR product and digest with MspI Cuts at CCGG Examine paoern of products to see how different the isolates are from the same food Want to sequence two different species

19 Different PCR products should give different MspI paoerns Panel I is an MspI digest of bacteria isolated from a sponge Image from hop://img.springerimages.com/images/springer /PUB=Springer_Netherlands Dordrecht/JOU=10482/VOL= /ISU=4/ART =2007_9169/MediaObjects/WATER_10482_200 7_9169_Fig1_HTML.jpg

20 Aier PCR, we will send 1 sample/group for sequencing, aier performing PCR clean up (both next week)