بعض تكتيكات RT PCR كتبتها أثناء مذاكرتي وكانت أمتع ماده بالنسبه ليا
Molecular (Genetics) 🧬🤗👍🏻
Molecular (Genetics) 🧬🤗👍🏻
▶️Why Should be converted RNA to cDNA 🤔??
Because the RNA is sensitive and easily destroy.
• Convert to cDNA to make it more stable to process of PCR.
Because the RNA is sensitive and easily destroy.
• Convert to cDNA to make it more stable to process of PCR.
▶️Differences between DNA and CDNA????
🧬 DNA is naturally found in our body and have coding Regions ( Exone) and Noncoding Regions( Interone ) that callude premature DNA
🧬 DNA is naturally found in our body and have coding Regions ( Exone) and Noncoding Regions( Interone ) that callude premature DNA
CDNA modified in invitro that converted RNA to CDNA by reverse transcriptase .
The cDNA is mature DNA not contains noncoding (intron ) only coding Regions
The cDNA is mature DNA not contains noncoding (intron ) only coding Regions
▶️Why we use RNA not DNA IN RT PCR???🤔
Because DNA not clear gene expression
But RNA highly Gene Expression Level
And same viruses genetic material is RNA type
Because DNA not clear gene expression
But RNA highly Gene Expression Level
And same viruses genetic material is RNA type
ايش الفرق بين PCR العادي و RT PCR ؟؟؟
الفرق انو pcr العادي يحتاج
ل Gel electrophoresis
عشان نشوف الباند او الرزلت
لاكن PCR العادي مايحتاج Gel electrophoresis لان كل الرزلت و النتائج راح اطلع لنا في الجهاز ع طول وراح نستخدم صبغه تظهر لنا النتائج ع طول 🤗
الفرق انو pcr العادي يحتاج
ل Gel electrophoresis
عشان نشوف الباند او الرزلت
لاكن PCR العادي مايحتاج Gel electrophoresis لان كل الرزلت و النتائج راح اطلع لنا في الجهاز ع طول وراح نستخدم صبغه تظهر لنا النتائج ع طول 🤗
الصبغات الا نستخدمها
▶️Type of fluorescent reporter
molecules ( fluresent dayes ) ???
1- SYBR green
2- sequence specific probes
Sequence spasific probes have 3 kind or type
⁃Taq Man Probes
⁃-Molecular Becons
⁃-Light cycler
▶️Type of fluorescent reporter
molecules ( fluresent dayes ) ???
1- SYBR green
2- sequence specific probes
Sequence spasific probes have 3 kind or type
⁃Taq Man Probes
⁃-Molecular Becons
⁃-Light cycler
▶️Intraclatire day SYBR Green is stine DNA binding Day Bind to minor groove ( stine dNTPs )
When bind withe dsDNA (dNTPs) متى تطلع ضوء
لما ترتبط بين القواعد النيتروجينيه
When bind withe dsDNA (dNTPs) متى تطلع ضوء
لما ترتبط بين القواعد النيتروجينيه
Cons??? سلبيات Non spasphic can lead to false positives can bind withe primer dimer
Pros ايجابيات??? Cheap don't require probe design
Directly probational When increase DNA products fluorescent increase
Pros ايجابيات??? Cheap don't require probe design
Directly probational When increase DNA products fluorescent increase
▶️ Sequence spasific probes
What is the probes 🤔???? Is particular sequence only bind to spasphic target sequence ( single strand DNA bind to spasphic complimentary strand that found in target DNA
تشبه البرايمر ماترتبط الا بسيكونس معين بس
What is the probes 🤔???? Is particular sequence only bind to spasphic target sequence ( single strand DNA bind to spasphic complimentary strand that found in target DNA
تشبه البرايمر ماترتبط الا بسيكونس معين بس
تتكون من جزئين اساسيين
Probes contain
1- 🌝💥 مضيئه مثل الشمسFlouriphore at 5’ end is molecule that emits light 💡 of certain wavelength
Emiats light at ground state the excess energy released 🔋
Probes contain
1- 🌝💥 مضيئه مثل الشمسFlouriphore at 5’ end is molecule that emits light 💡 of certain wavelength
Emiats light at ground state the excess energy released 🔋
2-🌚 معتمه مثل القمر لاكنها تمتص الضو من الشمس
Quencher at 3’ end is molecule that accepts energy from Fluorophore and dissipates energy either in the form of light or heat
Quencher at 3’ end is molecule that accepts energy from Fluorophore and dissipates energy either in the form of light or heat
😎 1- Taqman probes
Are hydrolysis probes that are designed to increase. The security of Quantitative pcr
How work??🤔
1- During annealing step probe attach to target strand downstream of the primre
Are hydrolysis probes that are designed to increase. The security of Quantitative pcr
How work??🤔
1- During annealing step probe attach to target strand downstream of the primre
2- both reporter and Quencher close to each other transfer energy 🔋 from reporter to Quencher there no signal
3- During extension step primer move to synthesis new strand the probe cleavage by 5’ exonuclease activity of Taq polymerase
3- During extension step primer move to synthesis new strand the probe cleavage by 5’ exonuclease activity of Taq polymerase
4- when operated the reborta emite florescent and day decrease in Quncher
5- signal captured by sequence Detection instrument and displayed by software
5- signal captured by sequence Detection instrument and displayed by software
🌸2- Molecular beacons :are single stranded hairpin shaped oligonucleotide probes. which contains complementary sequence to the target strand.
molecular beacon consists of 4 parts
•🌈Loop: contains 18-30 nucleotide which are complementary to the target sequence.
•🌈Loop: contains 18-30 nucleotide which are complementary to the target sequence.
•🌈Stem: The beacon stem sequence lies on both the ends of the loop
It is typically 5- 7 bp long at the sequences at both the ends are complementary to each other.
It is typically 5- 7 bp long at the sequences at both the ends are complementary to each other.
•🌈5' fluorophore (Reporter): At the 5' end is fluoresces day .
•🌈3' quencher (non fluorescent): The quencher dye is covalently attached to the 3’ end of the molecular beacon and when the beacon is in closed loop shape, it prevents the fluorophore from emitting light
How works ???🤔
The probe bind withe target sequence and become street open and bind to complementary strand and the florescent increase
ليش يطلع فلورسنت لان؟؟
Q and F ابتعدو عن بعض
The probe bind withe target sequence and become street open and bind to complementary strand and the florescent increase
ليش يطلع فلورسنت لان؟؟
Q and F ابتعدو عن بعض
🌸 Light cycler Hybridization probes
That arrangement Head to Tail .
🦋Two Hybridization probes
1- oligo 1 carries fluorescein label at 3’ , donar fluorescent
2- Oligo2 carries (LC Red 640 ) at 5’. Accept fluorescent
That arrangement Head to Tail .
🦋Two Hybridization probes
1- oligo 1 carries fluorescein label at 3’ , donar fluorescent
2- Oligo2 carries (LC Red 640 ) at 5’. Accept fluorescent
Principle
Depend on fluorescence Resonance Transfer (FRET)
what FRET is transport energy to ▶️
donar probe to ▶️ acceptor probe
Depend on fluorescence Resonance Transfer (FRET)
what FRET is transport energy to ▶️
donar probe to ▶️ acceptor probe
During annileing step primer and probes hybridize to specific target region
The machine give same signal to oligo1 Donar become active and emaite green fluorescent longer wavelength and then the acceptor emitted Red fluorescent
The machine give same signal to oligo1 Donar become active and emaite green fluorescent longer wavelength and then the acceptor emitted Red fluorescent
And then convert to signal read by computer 🤖
ان اصبت فمن الله وإن اخطأت فمني ومن الشيطان انا مجرد طالبه يارب اكون قدمت لو شي بسيط افيد فيه غيري 🙏🏼🤗
ان اصبت فمن الله وإن اخطأت فمني ومن الشيطان انا مجرد طالبه يارب اكون قدمت لو شي بسيط افيد فيه غيري 🙏🏼🤗
@Lolobushra1 بعد عمليه النسخ و الترجمه يظهر البروتين او التعبير الجيني بعد ما يتم اخذ المناطق الكود فقط
يعني من راح يطلع لنا تعبير جيني ؟RNA
هذا في الجسم
يعني من راح يطلع لنا تعبير جيني ؟RNA
هذا في الجسم
@Lolobushra1 في pcr ايش الا يحصل
انو عندنا RNA يعني مافيه Intone فيه بس only Exon يتم عكس التفاعل وتحويله cDNA هذا cDNA يكون خالي من Intron فيكون افضل في التعبير الجيني
يارب قدرت اوضح 🙏🏼🤗
انو عندنا RNA يعني مافيه Intone فيه بس only Exon يتم عكس التفاعل وتحويله cDNA هذا cDNA يكون خالي من Intron فيكون افضل في التعبير الجيني
يارب قدرت اوضح 🙏🏼🤗
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