Team:Cambridge/Protocols/PCRcolony

From 2012.igem.org

(Difference between revisions)
(Colony PCR:)
Line 3: Line 3:
==Colony PCR:==
==Colony PCR:==
-
 
+
Used to amplify DNA directly from cell culture, the reaction is the same as for normal PCR but with modified reaction composition and cycle settings, here shown for a 50 µl reaction using Taq polymerase.
-
<big>Used to amplify DNA directly from cell culture, the reaction is the same as for normal PCR but with modified reaction composition and cycle settings, here shown for a 50 µl reaction using Taq polymerase.</big>
+
{| class="wikitable" style="text-align: center; color: purple;"
{| class="wikitable" style="text-align: center; color: purple;"
Line 46: Line 45:
-
<big>'''Safety considerations:''' It is important to observe correct laboratory procedure and wear appropriate clothing and gloves. PCR occurs at high temperature and this may present a risk, depending on the PCR machine employed. For handling the cell culture, appropriate measures should be in place to deal with biohazardous waste.</big>
+
'''Safety considerations:''' It is important to observe correct laboratory procedure and wear appropriate clothing and gloves. PCR occurs at high temperature and this may present a risk, depending on the PCR machine employed. For handling the cell culture, appropriate measures should be in place to deal with biohazardous waste.

Revision as of 12:42, 7 August 2012

Colony PCR:

Used to amplify DNA directly from cell culture, the reaction is the same as for normal PCR but with modified reaction composition and cycle settings, here shown for a 50 µl reaction using Taq polymerase.

ReagentVolume (µl)Final Concentration
Water35.7
10 mM dNTPs1200 µM
10 x NH4 buffer51x
Forward Primer2.50.5 µM
Reverse Primer2.50.5 µM
Template Cells1.3 (from liquid culture or picked colony
Taq polymerase 5u/µl10.1 u/ µl



PCR machine settings:

Temperature (oc)Time (s)
Step 1 (cell breakage)95360
Step 2 (Cycle 30x)Denaturing9810
Annealing6030
Elongation72120
Step 3 (final extension)72300



Safety considerations: It is important to observe correct laboratory procedure and wear appropriate clothing and gloves. PCR occurs at high temperature and this may present a risk, depending on the PCR machine employed. For handling the cell culture, appropriate measures should be in place to deal with biohazardous waste.



Back to Protocols