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Questions and Answers
What is the primary function of phosphatases in the context of molecular cloning?
What is the primary function of phosphatases in the context of molecular cloning?
- To cleave 5' phosphate groups from nucleic acids. (correct)
- To add phosphate groups to the 3' hydroxyl ends of DNA.
- To protect DNA from degradation by nucleases.
- To catalyze the formation of phosphodiester bonds between DNA fragments.
Why is dephosphorylation of a vector plasmid often performed prior to ligation?
Why is dephosphorylation of a vector plasmid often performed prior to ligation?
- To prevent the vector from recircularizing without an insert. (correct)
- To make the plasmid more resistant to restriction enzyme digestion.
- To enhance the activity of the ligase enzyme.
- To increase the rate of the ligation reaction.
Which of the following enzymes is commonly used for dephosphorylation in molecular cloning?
Which of the following enzymes is commonly used for dephosphorylation in molecular cloning?
- Reverse transcriptase
- Restriction endonuclease
- Shrimp alkaline phosphatase (SAP) (correct)
- T4 DNA ligase
What chemical group is essential on the 5' end of a DNA fragment for effective ligation by T4 DNA ligase?
What chemical group is essential on the 5' end of a DNA fragment for effective ligation by T4 DNA ligase?
What is the role of T4 DNA ligase in DNA ligation?
What is the role of T4 DNA ligase in DNA ligation?
What two chemical groups are required for T4 ligase function during ligation?
What two chemical groups are required for T4 ligase function during ligation?
What is the role of ATP in a ligation reaction using T4 DNA ligase?
What is the role of ATP in a ligation reaction using T4 DNA ligase?
In the context of DNA ligation, what is 'recircularization'?
In the context of DNA ligation, what is 'recircularization'?
How does a low DNA concentration affect the outcome of a ligation reaction?
How does a low DNA concentration affect the outcome of a ligation reaction?
What is the purpose of using a 3:1 molar ratio of insert to vector in a ligation reaction?
What is the purpose of using a 3:1 molar ratio of insert to vector in a ligation reaction?
What would be the likely next step to do if a digestion did not work?
What would be the likely next step to do if a digestion did not work?
If redoing a digestion, what step in a typical protocol can be skipped?
If redoing a digestion, what step in a typical protocol can be skipped?
What is the purpose of ligating the lux operon from A. fischeri gDNA into a plasmid?
What is the purpose of ligating the lux operon from A. fischeri gDNA into a plasmid?
Which of the following is NOT a possible outcome of a ligation reaction between a plasmid and an insert?
Which of the following is NOT a possible outcome of a ligation reaction between a plasmid and an insert?
Besides recombination, what other process uses ligation in normal cells?
Besides recombination, what other process uses ligation in normal cells?
What is the final step after ligation and transformation?
What is the final step after ligation and transformation?
According to the notes, what should you do to a vial of ligase buffer taken out of the freezer?
According to the notes, what should you do to a vial of ligase buffer taken out of the freezer?
What ratio of insert to vector is considered a good starting point?
What ratio of insert to vector is considered a good starting point?
What should you always make sure of before leaving the lab?
What should you always make sure of before leaving the lab?
You perform a restriction digest and heat inactivate the enzyme. Do you need to perform this step again after dephosphorylation?
You perform a restriction digest and heat inactivate the enzyme. Do you need to perform this step again after dephosphorylation?
How long should a dephosphorylation reaction be performed?
How long should a dephosphorylation reaction be performed?
What is the formula given to calculate the required mass of insert?
What is the formula given to calculate the required mass of insert?
Given a 3:1 molar ratio, 1 g vector, and an insert to vector ratio of 900bp/300bp, what is the mass insert in grams?
Given a 3:1 molar ratio, 1 g vector, and an insert to vector ratio of 900bp/300bp, what is the mass insert in grams?
You have an insert at 20 g/mL and vector at 15 g/mL. For a ligation requiring 9g insert and 1g vector, how much insert and vector is required, respectively?
You have an insert at 20 g/mL and vector at 15 g/mL. For a ligation requiring 9g insert and 1g vector, how much insert and vector is required, respectively?
True or False: Double checking the math of ligation is generally considered difficult.
True or False: Double checking the math of ligation is generally considered difficult.
Which of the following alkaline phosphatases is NOT easily inactivated?
Which of the following alkaline phosphatases is NOT easily inactivated?
True or False: Phosphatases add 5' phosphates from nucleic acids.
True or False: Phosphatases add 5' phosphates from nucleic acids.
5' Phosphate is needed to ligate which end of another DNA strain?
5' Phosphate is needed to ligate which end of another DNA strain?
What is the molecular weight of T4 DNA ligase monomer?
What is the molecular weight of T4 DNA ligase monomer?
Flashcards
Dephosphorylation
Dephosphorylation
A process that removes 5' phosphates from nucleic acids, preventing self-ligation of plasmids.
DNA Ligases
DNA Ligases
Enzymes that join DNA strands by forming phosphodiester bonds.
T4 DNA Ligase
T4 DNA Ligase
Enzyme that requires 3' OH and 5' Phosphate to catalyze phosphodiester bonds.
Ligation
Ligation
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Low DNA concentration in ligation
Low DNA concentration in ligation
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High DNA concentration in ligation
High DNA concentration in ligation
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Insert: Vector Ratio
Insert: Vector Ratio
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DNA concentration
DNA concentration
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Study Notes
Dephosphorylation
- Phosphatases are used to cleave 5' phosphates from nucleic acids
- Common examples of phosphatases used include:
- Bacterial alkaline phosphatase
- Calf intestinal phosphatase (CIP)
- Shrimp alkaline phosphatase (SAP) is commercially available and easily inactivated
- This process reduces recircularization of plasmids during ligations
- 5' Phosphate is needed to ligate 3' OH
DNA Ligases
- Enzymes facilitate the joining of DNA strands
- Enzymes catalyze the formation of phosphodiester bonds between strands
- T4 DNA ligase (phage origin)
- Requires 3' OH and 5' Phosphate
- 487 amino acid monomer
- ATP dependent reaction
Ligation
- Ligation occurs often in normal cells
- Recombination occurs when Holliday junctions are filled in
- Replication occurs when Okazaki fragments are filled in
- It can be used for cloning
- Ligate the lux operon from A. fischeri gDNA into a plasmid
- Purpose: Put the recombinant plasmid into E. coli to express the non-native lux
Possible Outcomes of Ligation
- Insertion of lux operon
- Failure to ligate, linear DNA
- Insertion of another sequence from gDNA
- Recircularize to self
Effect of DNA Concentration
- Probability of ligation is related to the probability that the complementary DNA ends find each other.
- Low DNA concentration: Vector has a higher chance to recircularize to itself
- Easier to find the other end of itself than insert DNA
- Intramolecular ligation
- High DNA concentration: Dimers form (Concatemers)
- Easy for the ends to find each other between vector and insert (and maybe more insert!)
- Intermolecular ligation
DNA Concentration
- 3:1 molar ratio (insert:vector) is a good starting point to determine how much vector (plasmid) and how much insert (gDNA) is needed
- By adding more insert, there is a better chance it will get cloned into the vector instead of vector recircularizing
- Multiple ratios can be done to see what works best
Calculations
- required mass insert (g) = desired insert/vector molar ratio x mass of vector (g) x ratio of insert to vector lengths
- Example: (3/1 molar ratio) x 1g vector x (900bp insert/300bp vector) = mass insert (g)
- 3 * 1 * 3 = 9 g insert (per 1 g vector, for a 3:1 molar ratio)
Calculations Cont.
- Once you know how many micrograms you want, you need to figure out how many microliters of insert and vector to add to reaction:
- Lets say you have 20 g/mL insert and 15 g/mL vector:
- Using the above numbers: 9g insert/ 20g/mL = 0.45 mL
- 1g insert/ 15g/mL = 0.0667 mL
Experiment Notes
- Double check ligation math, it can be tricky
- Dephosphorylate for 20-30 minutes, not 10
- If you heat inactivated at the end of your digestion step, you do not need to do so again
Course Notes
- Open lab time
- Ensure to turn off gas and nanodrop, but do not turn off water baths
- Topic/Presentation paper choice is due 2/21/25
- Final paper introduction draft due 2/26/25
- Peer review due 3/5/25
- Quiz 3 is on 2/21/25
- Vortex ligase buffer, or anything coming out of the freezer!
- Important notes regarding tip boxes, freezer box labeling, and gel preparation
What to do if digestion didn't work?
- Backtracking:
- Redo digestion
- Redo plasmid and genomic DNA extraction
- Redo digestion using known workable DNA and/or plasmid
- If redoing digestion, tubes A and B can be skipped
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