PCR EtBr Gels Restriction Digest Ligation By Eunice Rhee and - - PowerPoint PPT Presentation

PCR EtBr Gels Restriction Digest Ligation

By Eunice Rhee and Christine Ahn

• I. Polymerase Chain Reaction

(PCR)

PCR: Why do we do it?

Developed in 1983 by Kary Mullis Amplify copies of DNA Also used in…

Sequencing of genes Identification of genetic fingerprints Diagnosis of diseases

PCR: How do we do it?

• 1. Denaturation of

double-stranded target DNA (by heat, 95°C)

• 2. Annealing of primers

to single-stranded DNA (by cooling, approx. 55°C)

• 3. Elongation of DNA

strands to make new DNA molecules (always 72°C)

PCR: Thermostable Polymerases

Taq DNA polymerase (first isolated from Thermus aquaticus, a hot springs bacterium) is a thermophilic DNA polymerase used because it can withstand high temperatures.

Maximal catalytic activity ranges around 75 to 80°C Magnesium ion is needed to shield negative charges

Pfu DNA polymerase (from Pyrococcus furiosus) seems to have the lowest error rate out of the thermophilic DNA polymerases

PCR: Procedure

• 1. Obtain forward and reverse primers
• 2. Prepare four reaction tubes:

Tube 1 – 0 ul Mg, 40.6 ul RNAse-free water Tube 2 – 1 ul Mg, 39.6 ul RNAse-free water Tube 3 – 2 ul Mg, 38.6 ul RNAse-free water Tube 4 – 3 ul Mg, 37.6 ul RNAse-free water

All tubes also contain 1ul forward primer, 1ul reverse primer, 5ul polymerase buffer, 1ul polymerase, 1ul template (i.e., E.Coli), and 0.6ul dNTPs

PCR: Procedure

• 3. Transfer each reaction tube mixture to PCR

tubes

• 4. Place tubes in thermocycler and set timer and

temperatures to allow DNA to denature, anneal, and elongate.

• II. Gel Extraction

and Ethidium Bromide Gels (EtBr)

Gel Extraction: Why do we do it?

Verify to check if you have the target DNA Separate target DNA from other strands or PCR products (purify DNA)

Gel Extraction: Procedure

• 1. Prepare DNA agarose gel and wells
• 2. Obtain tubes used for PCR
• 3. Add 10% of DNA SLB

(Ethidium Bromide dye)

• 4. Insert ladder in well 1
• 5. Fill next well with first tube, and proceed

the same way with the remaining tubes

• 6. Raise voltage to 100V for 25 minutes

Gel Extraction: Procedure

• 7. Observe where bands are

by comparing it to the ladder

• 8. Take photo of gel, then cut each band
• ut and place them each into separate 1g

microcentrifuge tubes and weigh them (to see how much QG to add)

• 9. Add QG buffer

(solubilization & binding buffer)

Gel Extraction: Procedure

• 10. Heat at 50°C for 15 minutes to allow agarose to melt.
• 11. Transfer to purple binding columns and centrifuge at

max speed for 1 min. (wash out QG)

• 12. Add wash buffer (PE) and centrifuge at max speed

for 1 min.

• 13. Dump out elute, then centrifuge for 1.5 min to

remove all ethanol

• 14. Transfer the filters to new centrifuge tubes and add

30ul of RNAse-free water

• 15. Centrifuge for 1 minute and keep the purified DNA

PCR Blunt

High copy vector Used for sequencing (make sure your insert is correct and present) Bidirectional Simple and fast!

1 2 Blunt ends 1 2 1 2 Direction does not matter!

1 uL salt sln 4 uL insert 1 uL PCR Blunt (15 min) (2 uL)

Send to sequencing…… If results are (+)……

Expression Vector

(pETM11)

Low copy Used for expression….protein analysis Restriction digest! Unidirectional Takes longer, more complex

Restriction Digest

10 uL pETM11 1 uL RE1 1 uL RE2 5 uL Buffer 33 uL RNase free H20 Total: 50 uL 40 uL Insert 1 uL RE1 1 uL RE2 3 uL Buffer 5 uL RNase free H20 Total: 50 uL

Digest O/N…

Restriction Enzymes

Available in our lab:

Nco I EcoR I Hind III BamH I Xho I Nde I

Palindromic sequences!