PCR and COLONY PCR
Introduction
Materials
- primers (stored in P1 39 -20°C lord of the ringsfridge (lotrf))
- plasmids (stored P1 39 -20°C fridge 1)
- 2mM dNTPs (stored in P1 38 freezer, 1 aliquot is stored in our drawer)
- HF buffer (stored in P1 38 freezer, 1 aliquot is stored in our drawer)
- X7 polymerase (red cap tube stored in P1 38 freezer)
- frozen rack or box full of ice to keep the enzymes cold (stored in P1 38 freezer)
- styropor box and crushed ice from coldroom
- minitubes for PCR
- 1.5mL Eppendorf tubes for mastermix and primermixes
Procedure
- look which primers to use with snapgene: look at plasmid map to check which primers are compatible
Prepare Mastermix
Ingredients are given for one reaction-> calculate how much you need for a specific number of reactions. Do the Mastermix on Ice and only when you're about to just use it.
ingredient ingredient | amount 1 X amount 1 X | amount 4X amount 4X | 5X 5X | x7 x7 | 10x 10x | 13X 13X | x15 x15 | 18x 18x | 20X 20X | 59 59 | |
1 1 | Buffer HF: | 5 | 20 | 25 | 35 | 50 | 65 | 75 | 90 | 100 | 235 |
2 2 | dNTPs: | 2.5 | 10 | 12.5 | 17.5 | 25 | 32.5 | 37.5 | 45 | 50 | 117.5 |
3 3 | X7 Polymerase: | 0.5 | 2 | 2.5 | 3.5 | 5 | 6.5 | 7.5 | 9 | 10 | 23.5 |
4 4 | Sterile MQ: | 14.75 | 59 | 73.75 | 103.25 | 147.5 | 191.75 | 221.25 | 265.5 | 295 | 693.25 |
NOTE : Sterile MQ: add up to 25 microliter: 25 -all the Mastermix ingredients - 1.25 (primers F and R mix)-1 (template) =14.75 μL for 1 reaction
- add 22.75 μL Mastermix to each strip, store this still on ice!
Prepare Primermix
A A | B B | |
1 1 | F primer | 4 μL |
2 2 | R primer | 4 μL |
3 3 | sterile MQ | 32 uL |
- add 1.25 μl Primermix to the appropriate microstrip
- Template DNA needs to be diluted: from minipreped overnight culture or directly colony PCR
MINIPREPED:
dilute 1 μl template in 9 μl MQ water
COLONY DILUTION:
With a pipette tip scrap off one colony only and add 10μl of MQ to corresponding labelled tube.
Remember that when we're doing colony PCR: streak out the rest of your diluted colony on a new plate so you have something if the colony PCR is positive, and mark with a number directly on the original plate which colony you picked!
- Add 1μl of colony dilution /template dilution to the appropriate microstrip
PCR machine (P1 28)
- Log in to our iGEM account, check if correct program, else adjust (samplevolume, tempetrature). Note: we need to book the thermocycler in the agenda beside it.
- Our program is called: X753 for short inserts, X7long for long inserts (FLS2, EFR, BAK) and X7long50 for different lower annealing temp (for FLS2+).
- Place stripps evently dispersed, if not enough use the green quadrat to ensure that the PCR machine doesn't close at a angle.
- COLONY PCR: same manner as outlined in the normal PCR, with the exception of starting the PCR with 6min at 95°C. The Program here is named COLONYLONG.
95ºC for 6 minutes
30x [95ºC for 30 sec,55ºC for 30 sec, 72ºC for 1kb/min]
72ºC for 20:00 min
Hold at 4ºC
- To confirm what our colony PCR product is, we do a Gel as always. Here are the ladders:
(Note: For LII constructs we do not need to amplify the whole sequence, a part of it is enough, since this will still allow us to successfully screen several different colonies quickly, as colony PCR with bigger fragements tend to be trickier.