Team:iTesla-SoundBio/LigatingofDigestedBackboneandInsertProtocolTemplate

Highlighted text are things that you need to fill in or change for your particular lab notebook depending on the variables, and concentrations.

 

Ligation of digested backbone and digested insert:

***To figure out volume of reactants in ligation reactions:***

1. Measure the concentration of the digested backbone in the nanodrop.
2. Using the NEBiocalculator, enter the insert length and vector length, and the vector mass that we want.
1. For example:
1. Insert Length (FC 1) → 1482 bp
2. Vector Length → 2114 bp (psb1a3), 2070 bp (psb1c3)
3. Vector Mass → 50 ng, vector volumn 50ng / conc
4. Insert Mass
3. Find the required mass of the insert that maintains a 3 :1 ratio (insert to vector).
4. Divide the required insert mass by the insert concentration to get the volume of insert that you need.
5. To find the volume of vector, take what you used for the vector mass (50 ng) and divide by the vector concentration to get the vector vector volume that you need.

 

Materials:

- digested insert [        ]

-gel extracted vector [             ]

- pcr tubes [Sound Bio storage]

-10X T4 DNA Ligase buffer [SoundBio Enzymes, Frosty]

-Ligase Enzyme [SoundBio Enzymes, Frosty]

-Rnase free water [wooden shelves, SoundBio storage]

-P10 pipette and tips

-pcr box tube rack

 

*Note: Keep the Ligase Buffer and Enzyme in freezer up until the moment you need it, both are very volatile*

 

________Trials 1-_: (_ tubes total of this reaction)

Psb1c3 #2 + Frag 1

- ___ ul H2O -___ ul vector -___ul insert -1 ul Buffer -1 ul Ligase Enzyme

Control:

______  Control

-__ ul H2O -__ ul vector -1 ul Ligase Buffer -1ul Ligase Enzyme

1. Label pcr tubes _______________
2. Add the reagents above in the order in which they appear.

3. Spin pcr tubes down for 5 seconds
4. Incubate at room temp for at least 10 minutes before continuing on to transformations.