[V] Ligation of the PCR product with the vector
[A] First, a small gel is run to determine concentrations of vector/insert to add to reaction
[B] Ligation Reaction:
Rxn1 Negative
Vector 2.0 2.0
Insert x --- Insert vol. based on calculation
Buffer y y 2x Buffer. Adjust
Ligase 1.0 1.0
H2O z z Adjust
Incubate at 25°C for 15 minutes
[C] Notes
[1] Negative control used to check transformation efficiency
[2] Incubate on PCR block
[3] Buffer volume calculated to the closest whole number after addition of vector, insert,
and ligase. E.g. Vector+insert+ligase = 8 uL therefore add 8uL buffer but no H2O.
[4] Vector is the cut p1.3 plasmid
[VI] Transformation of the Ligation
[A] After ligation has completed, proceed to transform the plasmid into competent E. coli cells
for cloning plasmid
[B] Obtain competent cells from -80°C freezer and keep on ice at all times
[C] Do all steps on ice. Avoid sudden temperature changes. Chill the pipet tips and
1.5mL tubes on ice.
[D] Protocol
[1] Add 3 uL ligation with 30 uL
[2] Mix by gently finger-flicking. Avoid spreading heat from fingers onto tubes; tap with
your nail.
[3] Incubate on ice 30 minutes.
[4] Heat-shock on 42°C for exactly 30 seconds. Critical step.
[5] Incubate on ice for 2 minutes.
[6] Add 300 uL LB and place in 37°C shaker for 45 minutes.
[7] Plate cells on LB-agar+Ampicillin plates
[8] Incubate overnight.
OTHER NOTES:
Preparing Ampicillin 1000x stock
[A] 100 mg/mL in H2O
[B] Protocol
[1] Dissolve 1g Amp sodium salt into 10 mL ddH2O (pure H2O)
[2] Aliquot into 10 tubes 1mL of the solution; store -20°C
[3] Use at 1:1000 dilution
Preparing LB Plates
[A] Put 4g LB_Agar in 500mL flask
[B] Add 100 mL H2O
[C] Microwave until powder dissolved. Watch for boilovers
[D] Cool to ~55°C
[E] Add 100uL of the 100mg/mL Ampicillin (at 1:1000 dilution)
[F] Mix; Pour 10-15mL into plates; Makes 7-8 plates
[G] Wrap, keep at 4°C
Preparing Lysogeny Broth
[A] Heat flask with 100mL H2O for 1 to 2 minutes to boil water
[B] Add 2g LB-POWDER to the water; it will dissolve automatically; some slight swirling will help
[C] Cool down and store 4°C
[1] DON'T add Ampicillin.
I have attached some notes I keep of transformation. In the end, I
added some preparation protocols. Hope it helps. It goes from ligation
to transformation.
On Thu, Mar 1, 2012 at 4:04 PM, Nathan McCorkle <nmz787@gmail.com> wrote:
>
> On Mar 1, 2012 1:58 PM, "Mega" <masterstorm123@gmail.com> wrote:
>>
>> reg. the plates:
>> http://www.benchfly.com/video/164/how-to-pour-lbagar-plates/
>> This video was very helpful (second vid)
>>
>>
>>
>> Thank you for your help!!
>>
>> Well, I could either dose the LB medium as it is said on the LB-Agar
>> package:
>> 10g LB-Agar per liter. I don't heat it, so the agar will sink to the
>> ground. The supernatant is then LB-water.
>>
>> Or I just use 'a lot' of LB-Agar (without calculating) and add some
>> water. What will stay at the ground of the tube is then agar mixed
>> with undissolved LB. (Not efficient, but for very small dosages maybe
>> ok)
> Huh?
>
> You need to heat agar, or it won't dissolve and would be pointless to add.
> Don't use too much or the salt concentration will be wrong, why wouldnt you
> just use what the package specifies?
>
> Or, are you saying you don't have LB without agar already added?
>
>>
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