IGEM:MIT/2007/Notebook/2007-6-13

Grad Advisors

 * Morning: Eric
 * Afternoon: L-A

=LAB WORK=
 * Purify digests on gel (EGFP and NNX)
 * Ligation overnight


 * Pour LB/Amp plates

Purifying the DNA
____  ____  ____  ____  ____  ____  ____  ____ 1V     1I    2V     2I    3V    3I    U      L   dd           dd            d xBa1         xBa1         xBa1 EcoR1       EcoR1
 * 1) Denature the enzymes by exposing the samples to high heat in the Master Gradient Cycler.
 * 2) Put the samples in, close lid
 * 3) Put 65°C for 15 minutes
 * 4) Put heat on and press enter
 * 5) Remember to turn off the heat when done!
 * 6) Prepare the gel
 * 7) Create a gel as usual (50 ml of Agar stuff, 5 µl of SYBR Safe DNA Stain)
 * 8) Put these ingredients together
 * 9) *25 µl of digest
 * 10) *6 µl of LB
 * L (Ladder)
 * 1kb Ladder  2µl
 * Loading Buffer 6 µl
 * H20    22 µl
 * Total: 30 µl because 1kb ladder is 500 ng/µl
 * U (Undigested)
 * 5 µl DNA
 * 6.25 µl Loading Buffer
 * 19 µl H20
 * Total: 30.25


 * Gel run at 85 volts for 45 min

Protocol for Purifying DNA from Agarose Gel

 * 1) Remove DNA fragment from agarose gel with razor blade
 * 2) Weigh slice in colorless tube, add 3 volumes Buffer QG or QX1 to 1 volume of gel (100mg ~ 100µl)
 * 3) Dissolve gel by incubate at 55°C temp block. vortex every 3 minutes until dissolved
 * 4) Make sure solution is yellow, if not consult Qiagen guidebook
 * 5) To bind DNA apply sample to a spin column and centrifuge for 1 min
 * 6) (Optional) Discard flow through and add .5ml Buffer QG and centrifuge for 1 min.
 * 7) To wash, add .75ml of PE Buffer and centrifuge for 1 min
 * 8) Centrifuge again to spin off excess
 * 9) Place column in clean centrifuge tube
 * 10) To elute, add 50 µl H20 and centrifuge for 1 min

Ligation
Reactants		ddV		ddV+ddI		dV		dV+dI Vector			11 µl		11 µl		4 µl		4 µl Insert			-		3 µl		-		2.5 µl 10x Ligation Buffer	2 µl		2 µl		2 µl		2 µl H2O			6.5 µl		3.5 µl		13.5 µl	       11 µl T4 Ligase		.5 µl		.5 µl		.5 µl		.5 µl Total			20 µl		20 µl		20 µl		20 µl
 * Best ratio is between 3:1 to 5:1 of insert to backbone
 * Use this equation
 * $$M_{I} = Moleratio_{\frac{I}{v}} * \frac{bp_{I}}{bp_{v}} * M_{vector}$$
 * Usually want $$M_{v}$$ to be between 20 ng and 100 ng
 * Once we solve for $$M_{i}$$ we should multiply it by between 3-5 to get the appropriate ratio
 * Actual Calculation for a 4:1 ratio
 * We decide $$m_{v}$$ = 40 ng and rearrange the equation.
 * $$\frac{4695}{720}=\frac{m_{v}}{m_{I}}*4$$
 * $$m_{I} = 24.61 ng$$
 * Reagent List for the Ligations
 * ddV:Double Digested Vector
 * ddI:Double Digested Insert
 * dV:Single Digested Vector
 * dI:Single Digested Insert
 * Reagent Amounts
 * Insert the ligations into a PCR machine and run the IGLIGATE Program
 * IGLIGATE -> Tubes -> Volume = 20 µl
 * 16°C for 5 hours
 * 60°C for 10 minutes to heat kill the enzymes

Second Double Digestion
7.5 µl		NNX 2.5 µl		NEB Buffer 2 2.5 µl		10x BSA .5 µl		EcoR1 .5 µl		Xba1 11.5 µl 	H20 Total		25 µl
 * We are making more double digested NNX vector (ddNNX)
 * $$\frac{1.5 g}{.204 ng/\mu l} = 7.35 \mu l$$
 * dd Mixture
 * Set at 37°C for 1 hour
 * Put at 65°C for 20 min to kill restriction enzymes

Making LB Amp Agar Plates

 * 1) Obtain LB from stock room in far side of 5th floor
 * 2) Microwave at 50% power for 10 minutes
 * 3) Remove from microwave; it should be boiling and smell like wet dog
 * 4) Place in 55°C water bath for 5-10 minutes
 * 5) Wait until it is cool to the touch
 * 6) Add 500 µl of Amp
 * 7) Swirl Gently
 * 8) *Avoid bubbles
 * 9) *Can quickly flame to remove them
 * 10) Mark the Plates with a Marker or Tape
 * 11) Pour and fill to 75% each plate
 * 12) Lift up lid
 * 13) Pour to cover ~90%
 * 14) Gently lift up plate and remove from stack
 * 15) If has bubbles, set aside for removal of bubbles
 * 16) Remove the bubbles
 * 17) Can only be done on liquid state ones
 * 18) Take a Bunsen burner and quickly flame
 * 19) Let Cool
 * 20) If excess LB remains, let harden then remove and throw away
 * 21) *Don't pour it down the sink!

Second Electrophoresis of Digestion
 Ladder		AL		JCH 1kb		ddNNX		ddNNX from 6.12 #2	from 6.12 #3 1 kb ladder	2 µl		-		- ddNNX		-		25 µl		25 µl Loading Buffer	6 µl		6 µl		6 µl H2O		22 µl		31 µl		31 µl