IGEM:MIT/2007/Notebook/2007-6-13
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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
- Denature the enzymes by exposing the samples to high heat in the Master Gradient Cycler.
- Put the samples in, close lid
- Put 65°C for 15 minutes
- Put heat on and press enter
- Remember to turn off the heat when done!
- Prepare the gel
- Create a gel as usual (50 ml of Agar stuff, 5 µl of SYBR Safe DNA Stain)
- 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
- 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
- Remove DNA fragment from agarose gel with razor blade
- Weigh slice in colorless tube, add 3 volumes Buffer QG or QX1 to 1 volume of gel (100mg ~ 100µl)
- Dissolve gel by incubate at 55°C temp block. vortex every 3 minutes until dissolved
- Make sure solution is yellow, if not consult Qiagen guidebook
- To bind DNA apply sample to a spin column and centrifuge for 1 min
- (Optional) Discard flow through and add .5ml Buffer QG and centrifuge for 1 min.
- To wash, add .75ml of PE Buffer and centrifuge for 1 min
- Centrifuge again to spin off excess
- Place column in clean centrifuge tube
- 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
- [math]\displaystyle{ M_{I} = Moleratio_{\frac{I}{v}} * \frac{bp_{I}}{bp_{v}} * M_{vector} }[/math]
- Usually want [math]\displaystyle{ M_{v} }[/math] to be between 20 ng and 100 ng
- Once we solve for [math]\displaystyle{ M_{i} }[/math] we should multiply it by between 3-5 to get the appropriate ratio
- Actual Calculation for a 4:1 ratio
- We decide [math]\displaystyle{ m_{v} }[/math] = 40 ng and rearrange the equation.
- [math]\displaystyle{ \frac{4695}{720}=\frac{m_{v}}{m_{I}}*4 }[/math]
- [math]\displaystyle{ m_{I} = 24.61 ng }[/math]
- Reagent List for the Ligations
- ddV
- Double Digested Vector
- ddI
- Double Digested Insert
- dV
- Single Digested Vector
- dI
- Single Digested Insert
- Reagent Amounts
<html><pre> <b>Reactants ddV ddV+ddI dV dV+dI</b> <b>Vector</b> 11 µl 11 µl 4 µl 4 µl <b>Insert</b> - 3 µl - 2.5 µl <b>10x Ligation Buffer</b> 2 µl 2 µl 2 µl 2 µl <b>H2O</b> 6.5 µl 3.5 µl 13.5 µl 11 µl <b>T4 Ligase</b> .5 µl .5 µl .5 µl .5 µl <b>Total</b> 20 µl 20 µl 20 µl 20 µl </pre></html>
- 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)
- [math]\displaystyle{ \frac{1.5 g}{.204 ng/\mu l} = 7.35 \mu l }[/math]
- dd Mixture
<html><pre> 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 <b>Total 25 µl</b> </pre></html>
- Set at 37°C for 1 hour
- Put at 65°C for 20 min to kill restriction enzymes
Making LB Amp Agar Plates
- Obtain LB from stock room in far side of 5th floor
- Microwave at 50% power for 10 minutes
- Remove from microwave; it should be boiling and smell like wet dog
- Place in 55°C water bath for 5-10 minutes
- Wait until it is cool to the touch
- Add 500 µl of Amp
- Swirl Gently
- Avoid bubbles
- Can quickly flame to remove them
- Mark the Plates with a Marker or Tape
- Pour and fill to 75% each plate
- Lift up lid
- Pour to cover ~90%
- Gently lift up plate and remove from stack
- If has bubbles, set aside for removal of bubbles
- Remove the bubbles
- Can only be done on liquid state ones
- Take a Bunsen burner and quickly flame
- Let Cool
- If excess LB remains, let harden then remove and throw away
- Don't pour it down the sink!
Second Electrophoresis of Digestion
<html><pre><b> Ladder AL JCH 1kb ddNNX ddNNX from 6.12 #2 from 6.12 #3</b> <b>1 kb ladder</b> 2 µl - - <b>ddNNX</b> - 25 µl 25 µl <b>Loading Buffer</b> 6 µl 6 µl 6 µl <b>H2O</b> 22 µl 31 µl 31 µl </pre></html>