IGEM:MIT/2007/Notebook/2007-6-13: Difference between revisions

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*Best ratio is between 3:1 to 5:1 of insert to backbone
*Best ratio is between 3:1 to 5:1 of insert to backbone
*Use this equation
*Use this equation
<pre>
**<math>M_{I} = Moleratio_{\frac{I}{v}} * \frac{bp_{I}}{bp_{v}} * M_{vector}</math>
M_insert = (mole_ratio_of_I:V) * (bp_I / bp_v) * M_vector
**Usually want <math>M_{v}</math> to be between 20ng and 100ng  
Usually want M_vector to be between 20ng and 100ng  
**Once we solve for <math>M_{i}</math> we should multiply it by between 3-5 to get the appropriate ratio
Once we solve for M_Insert we should multiply it by between 3-5 to get the appropriate ratio
</pre>
 
*Actual Calculation for a 4:1 ratio
*Actual Calculation for a 4:1 ratio
<pre>
<pre>
Line 69: Line 66:
m_I = 24.61 ng
m_I = 24.61 ng
</pre>
</pre>
<math>\frac{4695}{720}=\frac{m_{v}}{m_{I}}</math>
* Reagent List for the Ligations
* Reagent List for the Ligations
;ddV:Double Digested Vector
;ddV:Double Digested Vector
Line 75: Line 73:
;dI:Single Digested Insert
;dI:Single Digested Insert
* Reagent Amounts
* Reagent Amounts
<html>
<html><pre>
<pre>
<b>Reactants ddV ddV+ddI dV dV+dI</b>
<b>Reactants ddV ddV+ddI dV dV+dI</b>
<b>Vector</b> 11 ul 11 ul 4 ul 4ul
<b>Vector</b> 11 ul 11 ul 4 ul 4ul
Line 84: Line 81:
<b>T4 Ligase</b> .5 ul .5 ul .5 ul .5 ul
<b>T4 Ligase</b> .5 ul .5 ul .5 ul .5 ul
<b>Total</b> 20 ul 20 ul 20 ul 20 ul
<b>Total</b> 20 ul 20 ul 20 ul 20 ul
</pre>
</pre></html>
</html>
* Insert the ligations into a PCR machine and run the IGLIGATE Program
**IGLIGATE -> Tubes -> Volume = 20 ul
**16 degrees C for 5 hours
**60 degrees C for 10 minutes to heat kill the enzymes

Revision as of 11:16, 13 June 2007

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 degrees 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 ul of SYBR Safe DNA Stain)
    2. Put these ingredients together
      • 25 ul of digest
      • 6 ul of LB
____   ____  ____  ____  ____  ____  ____  ____ 
1V     1I    2V     2I    3V    3I    U      L   
dd           dd            d
xBa1         xBa1         xBa1
EcoR1        EcoR1
Figure 1: Gel Product
  • L (Ladder)
    • 1kb Ladder 2ul
    • Loading Buffer 6 ul
    • H20 22 ul
    • Total: 30 ul because 1kb ladder is 500 ng/ul
  • U (Undigested)
    • 5 ul DNA
    • 6.25 ul Loading Buffer
    • 19 ul 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 ~ 100ul)
  3. Dissolve gel by incubate at 55 degree 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 ul 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 20ng and 100ng
    • 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 m_v = 40 ng and rearrange the equation.
4695/720 = m_v / m_I * 4
m_I = 24.61 ng

[math]\displaystyle{ \frac{4695}{720}=\frac{m_{v}}{m_{I}} }[/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 ul 11 ul 4 ul 4ul <b>Insert</b> - 3 ul - 2.5 ul <b>10x Ligation Buffer</b> 2 ul 2ul 2ul 2 ul <b>H2O</b> 6.5 ul 3.5 ul 13.5 ul 11 ul <b>T4 Ligase</b> .5 ul .5 ul .5 ul .5 ul <b>Total</b> 20 ul 20 ul 20 ul 20 ul </pre></html>

  • Insert the ligations into a PCR machine and run the IGLIGATE Program
    • IGLIGATE -> Tubes -> Volume = 20 ul
    • 16 degrees C for 5 hours
    • 60 degrees C for 10 minutes to heat kill the enzymes
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