primer sense. - 3 primer antisense 5 - TAC AAG TAC TCA GAT CTC GAG CTC AAG CTT CGA ATT CNN NNN NNN NNN ATG NNN

Transcription

1 Activity that you must complete by Sunday November 11 th at 12 EGFP TAC AAG TAC TCA GAT CTC GAG CTC AAG CTT CGA ATT CTG CAG TCG restriction enzyme S restriction enzyme A restriction enzyme S restriction enzyme A - 3 primer sense - 3 primer antisense TAC AAG TAC TCA GAT CTC GAG CTC AAG CTT CGA ATT CNN NNN NNN NNN ATG NNN EGFP linker NRG1

2 5 -CGTTAACTTGACCATGTGCATCTAGCTCCATGGCATGC-3 5 -CGTTAACTTGACCATGTGCATCTAGCTCCATGGCATGC-3 3 -GCAATTGAACTGGTACACGTAGATCGAGGTACCGTACG-5 Primer sense: 5 -CGTTAACTTG-3 Primer antisense: 5 -GCATGCCATG-3 Added the HindIII site 5 -AAGCTT-3 to the 5 of the primer 3 -TTCGAA-5 sense Added the PstI site 5 -CTGCAG-3 to the 5 of the primer 3 -GACGTC-5 antisense Primer sense: 5 -AAGCTTCGTTAACTTG-3 Primer antisense: 5 -CTGCAGGCATGCCATG-3 5 -AAGCTTCGTTAACTTGACCATGTGCATCTAGCTCCATGGCATGC-3 3 -GCAATTGAACTGGTACACGTAGATCGAGGTACCGTACGGACGTC-5

3 5 -AAGCTTCGTTAACTTG-3 -> 3 -GCAATTGAACTGGTACACGTAGATCGAGGTACCGTACG-5 5 -CGTTAACTTGACCATGTGCATCTAGCTCCATGGCATGC-3 <- 3 -GTACCGTACGGACGTC-5 5 -AAGCTTCGTTAACTTGACCATGTGCATCTAGCTCCATGGCATGC-3 3 -GCAATTGAACTGGTACACGTAGATCGAGGTACCGTACGGACGTC-5 5 -AAGCTTCGTTAACTTGACCATGTGCATCTAGCTCCATGGCATGCCTGCAG-3 3 -TTCGAAGCAATTGAACTGGTACACGTAGATCGAGGTACCGTACGGACGTC-5

4 HindIII sense primer antisense primer PstI PRIMERS Sense: 5 -AAGCTTGGAGGTGAGCCGATGG-3 Antisense: 5 -CTGCAGCTACTCAGGCAGAGACAGAAAG-3 HindIII TRANSLATION PstI

5 RT-PCR amplification 1 - to verify/quantify the expression of a gene/specific isoform 2 - to clone the full length cdna to express the protein

6 1 - Primers on different exons separated by a big intron ( 1000 bp) Genomic DNA Promoter mrna cdna Exon1 Exons 2,3... Intron RNA polimerase RNA PCR RNA splicing polia RT (reverse transcriptase)

7 2 - Primers straddling two exons Genomic DNA Promoter mrna cdna Exon1 Exons 2,3... Intron RNA polimerase RNA RNA splicing PCR polia RT (reverse transcriptase)

8 Primers to verify & quantify the expression of specific isoforms Genomic DNA EGF-like α β Transcription (RNA polimerase) RNA splicing RT (reverse transcriptase) PCR EGF-like α EGF-like β

9 RNA extraction RNA quantification by spectrofotometer by Nanodrop Solo per uso didattico - vietata la riproduzione o la vendita

10 stock 1 sample final concentration RNA 0.1 µg/ µl µl 1 µg/reaction Buffer 5x µl 1x BSA 1 µg/µl µl 0.1 µg/ µl triton 1% µl 0.05% dntps 10mM µl 500 µm random primers 50 µm µl 5 µm RT 100u/µl µl 200u/reaction RNAsin 33u/µl µl 33u/reaction Water to 25µl µl Tot 25 µl

11 Dilution factor Concentration of the stock Final concentration NaCl 5M 0,5 M Total volume 25 µl How many fold do I have to dilute this ingredient?

12 Dilution factor Concentration of the stock Final concentration NaCl 5M 0,5 M Volume totale 25 µl How many fold do I have to dilute this ingredient? 5:0,5 = 10 fold = dilution factor The final volume is 25 µl, how many µl do I have to use?

13 Dilution factor Concentration of the stock Final concentration NaCl 5M 0,5 M Total volume 25 µl How many fold do I have to dilute this ingredient? 5 : 0,5 = 10 fold = dilution factor The final volume is 25 µl, how many µl do I have to use? I have to dilute 10 fold this ingredient in the final volume of the solution I will have to add 1/10 of the final volume 25 µl : 10 = 2,5 µl

14 stock 1 sample final concentration RNA 0.1 µg/ µl µl 1 µg/reaction Buffer 5x µl 1x BSA 1 µg/µl µl 0.1 µg/ µl triton 1% µl 0.05% dntps 10mM µl 0,5mM random primers 50 µm µl 5 µm RT 100u/µl µl 200u/reaction RNAsin 33u/µl µl 33u/reaction Water to 25µl µl Tot 25 µl

15 Contaminations RNA can be contaminated by genomic DNA if you put primers on different exons separated by a big intron, genomic contamination is not a problem BUT your gene of interest can have only one exon your gene of interest can have only small introns your gene of interest can present pseudogenes (what is a pseudogene?) How can you detect the presence of genomic DNA contamination, when you have single exons, small introns or pseudogenes? RNA or RT or PCR ingredients can be contaminated by DNA (plasmidic DNA or previous PCR products) How can you detect the contamination of plasmidic DNA or previous PCR products?

16 Pseudogenes are cdna copies inside genomic DNA. If you have genomic DNA contamination, you can amplify pseudogenes even if your primers are on different exons, separated by a big intron. Genomic DNA Promoter Exon1 Exons 2,3... Intron PSEUDOGENE RNA polimerase RNA RNA splicing mrna polya RT (reverse transcriptase) cdna PCR PCR!

17 Commonly used housekeeping genes are on a single exon or have pseudogenes

18 Commonly used housekeeping genes are single exon or have pseudogenes

19 GAPDH

20

21 GAPDH is not a good housekeeping gene, if your RNA is contaminated by genomic DNA!

22 !

23 Contaminations RNA can be contaminated by genomic DNA if you put primers on different exons separated by a big intron, genomic contamination is not a problem BUT your gene of interest can have only one exon your gene of interest can have only small introns your gene of interest can present pseudogenes (what is a pseudogene?) How can you detect the presence of genomic DNA contamination, when you have single exons, small introns or pseudogenes? RNA or RT or PCR ingredients can be contaminated by DNA (plasmidic DNA or previous PCR products) How can you detect the contamination of plasmidic DNA or previous PCR products?

24 RT Which control can you do to verify that RNA and the reverse transcription ingredients are not contaminated by DNA (genomic, plasmidic, amplification products)? stock 1 sample RT negative control final concentration RNA 0.1 µg/ µl µl 1 µg/reaction Buffer 5x µl 1x BSA 1 µg/µl µl 0.1 µg/ µl Triton 1% µl 0.05% dntps 10mM µl 0,5mM random primers 50 µm µl 5 µm RT 100u/µl µl 200u/reaction RNAsin 33u/µl µl 33u/reaction Water to 25µl µl Tot 25 µl 25 µl The negative control is called...

25 Reverse Transcription Tube 1 - RT on the sample/s that you have to analyse Tube 2 - control RT- : all the ingredients, except the reverse transcriptase (RT-)

26 PCR reaction Which control can you do to verify that the PCR reaction is not contaminated by DNA (plasmidic, amplification products)? STOCK sample # RT- PCR negative ctr final conc cdna 5 µl 5x buffer Taq µl -> 1x sense primer 10 M µl -> 250 nm antisense primer 10 M µl -> 250 nm 100% glycerol µl -> 5% 10mM dntps µl -> 100 µm Taq polimerase 1u/ul µl -> 1u H 2 O µl total 50 µl 50 µl

27 PCR reaction Which control can you do to verify that the PCR reaction is not contaminated by DNA (plasmidic, amplification products)? STOCK sample # RT - PCR negative ctr final conc cdna 5 µl 5 µl RT - 5x buffer Taq µl -> 1x sense primer 10 M µl -> 250 nm antisense primer 10 M µl -> 250 nm 100% glycerol µl -> 5% 10mM dntps µl -> 100 µm Taq polimerase 1u/ul µl -> 1u H 2 O µl total 50 µl 50 µl