IGEM:UC Berkeley/2006/Procedure Overview (Plasmid DNA to Sequencing)

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Current revision (14:35, 3 July 2006) (view source)
 
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2. Ligation<br<
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2. Ligation<br>
- DNA ligase connects double stranded fragements of DNA with reverse complement sticky ends<br>
- DNA ligase connects double stranded fragements of DNA with reverse complement sticky ends<br>
'''- Result: Relaxed double stranded circular DNA'''
'''- Result: Relaxed double stranded circular DNA'''

Current revision

1. Plasmid DNA
Path 1
- Restriction Digest of Plasmid DNA
- Run a Gel to separate parts
- Gel purify the desired part (not always going to be the shorter or longer band- could be either)
- Result: Double stranded linear DNA with sticky ends
Path 2
- PCR desired Plasmid
- Run an analytical gel to determine whether PCR worked correctly
- Result is liner double stranded DNA in buffer
- PCR cleanup
- Result is linear double stranded DNA in H2O
- Restriction Digest
- Cleanup- take only the strands that we want to insert into plasmid
- Result: Double stranded linear DNA with sticky ends


2. Ligation
- DNA ligase connects double stranded fragements of DNA with reverse complement sticky ends
- Result: Relaxed double stranded circular DNA


3. Transformation- insert new plasmid into cell
- Plate
- Observe results- are they expected or unusual?

4. Grow up colonies of cells in LB


5. Screen
Path 1
- Mini prep
- Restriction Map
- Result: Indentified clones
Path 2
- Colony PCR
- Result: Indentified clones

6. Sequence- is plasmid what it should be?
--Mfleming 14:34, 3 July 2006 (EDT)

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