How do you evaluate restriction site ends for compatibility?
compare opposite sides of each site The first four before cut off
Antisense stand
is used as a template to create a copy of the sense strand
Sense strand
used as a template to create a copy of the antisense strand
General rules for PCR primer design
1) you need BOTH a forward and reverse DNA primer
- forward primer same sequence as 5’-3’ of the sense strand
- reverse primer same sequence as 5’-3’ as the antisense strand
2) “ideally” PCR primers are -20 nucleotides long (range 18-30)
3) 50% A+T and 50% G+C( GC content usually ranges from 40-60%)
4) melting (annealing) temperature Tm=4(G+C) + 2(A+T) and usually around 60 degrees celsius
5) melting temperature of both primers needs to be close to each other (within 5 degrees celsius)
6) avoid secondary structures in your pimer sequence
What should be in your PCR reaction (in the tube)
- Template DNA (DNA you want to replicate)
- Forward DNA primer
- Reverse DNA primer
- dATP,dTTP,dGTP, dCTP (dNTPs)
- DNA polymerase (Taq)
- Magnesium, buffer
What are the typic steps for PCR reaction
- Denature 94 degrees celsius, 1 min
- Anneal **variable temp, 1 min
- Extend: 72 degrees celsius **variable time
Repeat steps 1-3 15-35 times
What is the rule of thumb for the amount of time for each kilobase
1 min
What does you need for maximum stringency?
optimal melting/annealing temp
What are the advantage of using a health liable Taq-specific antibody instead of standard “hot Start”
- reduces the risk of cross contamination bc it is not necessary to reopen the reaction tubes after heating
- can be used when other hot start methods are difficult to perform, such as high throughput PCR, in situ PCR, micro titer plate formats, capillary PCR, and oil free reactions
Pros of PCR cloning
- fast! PCR can be done in about 3 hours
- can generate ends appropriate for cloning without requiring restriction digestion of the product (blunt end or TA cloning)
- can purposefully incorporate mutations in the ends of the PCR products by engineering them into the primers (site directed mutagenesis)
Cons of PCR cloning
PCR can produce unwanted mutations (amplification in a test tube is more error prone than in vivo replication, in part bc purified polymerases have reduced proofreading ability)
- Primers can amplify the wrong target sequence on occasion, often due to priming at incorrect sites with similarity to the intended target sequence
- It is necessary to sequence clones derived from PCR-made inserts to ensure that there are no mistakes int he nucleic acid sequence
Steps for PCR cloning by incorporation of restriction sites?
- Decide which sites in your vector to clone into
- engineer those sites into your primers
- perform PCR as you would with restriction enzymes to create sticky ends
- Ligate PCR product (insert) into your vector
- screen for positive slopes
- sequence several positives to check for errors
