IGEM:MIT/2007/Notebook/2007-6-13: Difference between revisions
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*Gel run at 85 volts for 45 min | *Gel run at 85 volts for 45 min | ||
===Purifying DNA from Agarose Gel=== | ===Protocol for Purifying DNA from Agarose Gel=== | ||
#Remove DNA fragment from agarose gel with razor blade | #Remove DNA fragment from agarose gel with razor blade | ||
#Weigh slice in colorless tube, add 3 volumes Buffer QG to 1 volume of gel (100mg ~ 100ul) | #Weigh slice in colorless tube, add 3 volumes Buffer QG to 1 volume of gel (100mg ~ 100ul) | ||
#Dissolve gel by incubate at 55 degree C temp block. vortex every 3 minutes until dissolved | #Dissolve gel by incubate at 55 degree C temp block. vortex every 3 minutes until dissolved | ||
#Make sure solution is yellow, if not consult Qiagen guidebook | #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 ul H20 and centrifuge for 1 min | |||
==Ligation== | ==Ligation== | ||
Revision as of 08:10, 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
- Denature the enzymes by exposing the samples to high heat in the Master Gradient Cycler.
- Put the samples in, close lid
- Put 65 degrees 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 ul of SYBR Safe DNA Stain)
- 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
- 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
- Remove DNA fragment from agarose gel with razor blade
- Weigh slice in colorless tube, add 3 volumes Buffer QG to 1 volume of gel (100mg ~ 100ul)
- Dissolve gel by incubate at 55 degree 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 ul H20 and centrifuge for 1 min
Ligation
- Best ratio is between 3:1 to 5:1 of insert to backbone
- Use this equation
M_insert = (mole_ratio_of_I:V) * (bp_I / bp_v) * M_vector Usually want M_vector to be between 20ng and 100ng Once we solve for M_Insert 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
