Marianne and Vicki
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.
T4 DNA Ligase
Ligase buffer (1uL aliquots)
Insert and vector DNA (of approximately known concentration)
- 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.
- Add 1uL ligase buffer (vortex, and make sure it still smells like wet dog).
- Add diH2O to 9.5uL and vortex.
- Add 0.5uL T4 ligase.
- 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
|T4 DNA Ligase (add last)||1uL||1uL||1uL||1uL||1uL||1uL||1uL||1uL||1uL||1uL||1uL||1uL||12uL|
- 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)