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

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Contents

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

  1. Denature the enzymes by exposing the samples to high heat in the Master Gradient Cycler.
    1. Put the samples in, close lid
    2. Put 65°C for 15 minutes
    3. Put heat on and press enter
    4. Remember to turn off the heat when done!
  2. Prepare the gel
    1. Create a gel as usual (50 ml of Agar stuff, 5 µl of SYBR Safe DNA Stain)
    2. Put these ingredients together
      • 25 µl of digest
      • 6 µl of LB 
____   ____  ____  ____  ____  ____  ____  ____ 
1V     1I    2V     2I    3V    3I    U      L   
dd           dd            d
xBa1         xBa1         xBa1
EcoR1        EcoR1
First Gel Product
First Gel Product
  • 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

  • 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 Mv to be between 20 ng and 100 ng
    • Once we solve for Mi we should multiply it by between 3-5 to get the appropriate ratio
  • Actual Calculation for a 4:1 ratio
    • We decide mv = 40 ng and rearrange the equation.
    • \frac{4695}{720}=\frac{m_{v}}{m_{I}}*4
    • mI = 24.61ng
  • Reagent List for the Ligations
ddV
Double Digested Vector
ddI
Double Digested Insert
dV
Single Digested Vector
dI
Single Digested Insert
  • Reagent Amounts 

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

  • 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

  • We are making more double digested NNX vector (ddNNX)
  • \frac{1.5 g}{.204 ng/\mu l} = 7.35 \mu l
  • dd Mixture 

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

  • 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
    • Avoid bubbles
    • Can quickly flame to remove them
  8. Mark the Plates with a Marker or Tape
  9. Pour and fill to 75% each plate
    1. Lift up lid
    2. Pour to cover ~90%
    3. Gently lift up plate and remove from stack
    4. If has bubbles, set aside for removal of bubbles
  10. Remove the bubbles
    1. Can only be done on liquid state ones
    2. Take a Bunsen burner and quickly flame
  11. Let Cool
  12. If excess LB remains, let harden then remove and throw away
    • 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

Second Digestion Gel
Second Digestion Gel
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