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

iGEM Project name 1 Main project page
Previous entry      Next entry

## 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.
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
 PCR TUBE 1L 2L 3L 4L 5L 6L 7L 8L 9L 10L 11L 12L Total 1RD 0.310 0.310 2RD 0.310 0.310 3RD 0.310 0.310 4RD 0.310 0.310 5RD 0.310 0.310 6RD 0.310 0.310 7RD 0.310 0.310 8RD 0.310 0.310 9RD 0.310 0.310 10RD 0.310 0.310 11RD 0.310 0.310 12RD 0.310 0.310 psB1C3 5uL 5uL 5uL 5uL 5uL 5uL 5uL 5uL 5uL 5uL 5uL 5uL 60uL Ligase buffer 2uL 2uL 2uL 2uL 2uL 2uL 2uL 2uL 2uL 2uL 2uL 2uL 24uL dH2O 1.69uL 1.69uL 1.69uL 1.69uL 1.69uL 1.69uL 1.69uL 1.69uL 1.69uL 1.69uL 1.69uL 1.69uL 20.28uL T4 DNA Ligase (add last) 1uL 1uL 1uL 1uL 1uL 1uL 1uL 1uL 1uL 1uL 1uL 1uL 12uL Total 10uL 10uL 10uL 10uL 10uL 10uL 10uL 10uL 10uL 10uL 10uL 10uL
• 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)