Download Colony PCR from Yeast or Bacteria

Colony PCR from Yeast or Bacteria
Be sure to change your tip when handeling different templates/primers. Do
not contaminate reagents with other primers or template. Do not
contaminate samples with other tempate.
STEP 1: Making “dirty” template DNA.
In this step you will break open yeast/bacteria to liberate the DNA.
 Yeast
Add a swipe of a yeast colony to 50 uL of water containing 2 uL of lyticase in a 200
uL PCR tube. Be careful to get a single colony/patch (do not contaminate with
another colony/patch or with agar off the plate). Incubate at 37°C for 15 minutes,
then heat at 98°C for 10 minutes in the PCR machine.
 Bacteria
Add a swipe of a bacterial colony to 50 uL of water in a 200 uL PCR tube. Be careful
to get a single colony/patch (do not contaminate with another colony/patch or with
agar off the plate). Heat at 98°C for 5 minutes in the PCR machine.
STEP 2: REDtaq PCR
In this step you will amplify your gene of interest using REDtaq. REDtaq contains
gel loading buffer and will not interfere with the subsequent sequencing reaction. If
doing many reactions then make a master mix with everything but the template.
 Reaction mix
3.0 uL 10X REDTaq PCR Reaction Buffer
1 uL dNTP’s Mix
1 uL forward primer
1 uL reverse primer
1.25 uL REDTaq DNA polymerase (add last to mix)
1 uL template
22 uL H20
30 uL total
 PCR program
Step 1 (1 cycle):
Denature at 94°C for 2 min
Step 2 (33 cycles): Denature 94°C for 1 min
Anneal 55°C for 2 min
Extend at 72°C for 3 min
Step 3 (1 cycle):
Final extention at 72°C for 8 min
Step 4 (1 cycle):
Hold at 4°C forever
STEP 3: Visualize PCR on DNA gel
This step is required to verify the presence of PCR product.
STEP4: DNA sequencing reaction
This step will use fluorescent dNTP’s and a special polymerase for sequencing. SETUP THIS REACTION ON ICE. Make a master mix when appropriate.
 Reaction mix
2.0 uL of template (add to PCR tubes first, then add master mix)
1.0 uL of either the forward or the reverse primer
(1 primer only to amplify 1 DNA strand)
4.75 uL of water
2.0 uL of 5X buffer
0.5 uL of BigDye (contains fluorescent dNTP’s and the polymerase) *add last
10 uL total
 PCR program
STEP 1:
STEP 2:
STEP 3:
STEP 4:
When PCR finished, add 20 uL of water and place in the sequencing sample box. Be
sure to fill out the sequencing sheet on the front of the freezer.