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Sequencing

Analysis of Colonies and PCR II

1. Circle discrete colonies and circle them. Choose all white colonies and those that are light blue.

2. Make a table in order to track the colonies circled.

3. Summarize the colonies

a. If there are too few, go back to the original PCR product and replate; for example, we replated our D-5 plate and added 3 and 4ul of product instead of .1 and 2ul.
b. If there are no colonies, replate using 0.1 and 4ul of product.

4. Put a sample (using a pipette tip) of each colony chosen in the following cocktail:

PCR Cocktail

1ul Forward primer
2.5ul 10X buffer (with MgCl)
2.5ul DNTP
l7ul nH20
1ul Taq
25ul total

5. Place the reaction tubes in the PCR machine following protocol #25.