IGEM:UBC/2009/Notebook/UBC iGEM 2010/2010/06/24

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dspB Track

Marianne and Vicki

Ligations

Problem: XbaI and SpeI cut sites are complementary - same sticky ends, so there is a chance of dspB forming con-catemers with each other (forming a long chain). There is also a high chance of the backbone plasmid ligating back together without the insert.
Solution for now: continue with ligations and transformation. Need to colony PCR a LOT of colonies

Protocol: Refer to "iGEM common protocols". It is written below for easy access.
Supplies Needed: T4 DNA Ligase Ligase buffer (1uL aliquots) diH2O Insert and vector DNA (of approximately known concentration)

Steps:

  1. Calculate insert/vector amounts. Various ratios have been recommended, most commonly 3:1 or 6:1. Formula: insert mass (ng) = ratio x (insert length/vector length) x vector mass (ng)

Example: For a 150bp insert and a 2200bp vector, at a ratio of 6:1: insert mass = 9/22 * vector mass. If you have a measurement of 30ng/uL for the vector and 5ng/uL for the insert, you need 2.5uL of insert and 1uL of vector.

  1. Add 1uL ligase buffer (vortex, and make sure it still smells like wet dog).
  2. Add diH2O to 9.5uL and vortex.
  3. Add 0.5uL T4 ligase.
  4. Incubate at 37°C for at least 1 hour (leave overnight after transforming in case it needs to go for a bit longer).
  • Changes: Did an overnight ligation
    • Overnight ligation: Incubate at 37°C for half an hour, then put on ice and place in 4°C fridge.

Prior to ligations: inactivate enzymes by putting digests in 80ºC waterbath for 20 minutes.

Used the Nanodrop upstairs in Room 375? to determine the concentration of 7RD (H49-A).
[7RD] = 268.0ng/uL
*Assumption: Other DNA samples have similar concentration, to within +/- 10ng/uL
Used the Nanodrop upstairs to determine the concentration of psB1C3.
[psB1C3] = 11.7ng/uL

  • We want a ratio of 3:1
  • Insert mass = 16.58475 (using the formula in the protocol)
  • 1uL of vector : 0.0619uL of insert
    • Multiplied by 5uL because original values were too small for the Pipettman.
    • Therefore, pipet 5uL of vector to 0.310uL of insert
Table 1. Ligation Mixes
PCR TUBE1L2L3L4L5L6L7L8L9L10L11L12LTotal
1RD0.3100.310
2RD0.3100.310
3RD0.3100.310
4RD0.3100.310
5RD0.3100.310
6RD0.3100.310
7RD0.3100.310
8RD0.3100.310
9RD0.3100.310
10RD0.3100.310
11RD0.3100.310
12RD0.3100.310
psB1C35uL5uL5uL5uL5uL5uL5uL5uL5uL5uL5uL5uL60uL
Ligase buffer2uL2uL2uL2uL2uL2uL2uL2uL2uL2uL2uL2uL24uL
dH2O1.69uL1.69uL1.69uL1.69uL1.69uL1.69uL1.69uL1.69uL1.69uL1.69uL1.69uL1.69uL20.28uL
T4 DNA Ligase (add last)1uL1uL1uL1uL1uL1uL1uL1uL1uL1uL1uL1uL12uL
Total10uL10uL10uL10uL10uL10uL10uL10uL10uL10uL10uL10uL
  • Add 3.69uL of Ligation Master Mix (LMM) which includes ligase buffer and dH2O to each microcentrifuge tube 1L to 12L.

*To 10L, could not add 3.69uL because not enough was left. Added whatever was left - probably short 0.5uL to 1uL

Incubate in 37°C waterbath for half an hour, then placed on ice and left in 4°C fridge overnight.
Vicki Ma 04:43, 28 June 2010 (EDT)


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