Lab 18 PCR notes
Amplification of part of the l viral genome:
l is 48,502 bp long
PCR primers for this experiment are 20 nt long
Amplified fragment: 1,106 bp long
BamHI site: 22,346 (about 100 nt from the end)
HindIII site: 23,130 (about 200 nt from the end)
After digestion of the amplified DNA by BamHI & HindIII (Lab 19A):
784 bp fragment for cloning into pBLU
(new screening method for pBLU: not just antibiotic resistance, but “Blue/White” screening)
Lab 18A: PCR
94 C 1 min; 60 C 45 sec; 72 C 1 min; repeat 24 times; 4 C hold “181 lambda” program
Lab 18B: Purification of the PCR product. We will NOT use phenol/chloroform extraction of the proteins. Instead we will use commercially-produced spin columns.
This step is necessary because in lab 19 we will be cloning the PCR product into a plasmid. This cloning will use a sticky end ligation strategy. First, the PCR product will be digested with restriction enzymes (BamHI & HindIII) to generate sticky ends; then ligated appropriately. The PCR reaction itself contains not only the PCR product (DNA) but also surviving DNA polymerase, and remaining nucleotides. DNA polymerase can use these nucleotides to actually “fill in” the sticky ends created by the restriction enzymes (it can synthesize the complementary strand to the single-strand overhang, making the sticky end a blunt end). Spin column chromatography allows you to purify the DNA (PCR product) from the free nucleotides, enzyme, and PCR buffer.
Lab 18C: Understand the controls on this gel.
Lane 1 (PCR control): sample taken from PCR reaction BEFORE amplification. This is to confirm that the 1100 bp fragment was not already present before PCR.
Lane 2 (PCR product): sample taken from PCR reaction AFTER amplification but BEFORE purification. This is to confirm that PCR successfully amplified an 1100 bp DNA fragment.
Lane 3 (Purified PCR product): sample taken AFTER purification. This lane confirms that you did not lose your DNA during purification (not a rare event!).
QIAquick spin columns:
Column binds DNA 100 bp-10kb
“5 volumes” (measure). Save an aliquot before purification.
LEAVE mineral oil (top phase)
Resuspend/elute into 50 uL.
DO NOT add loading buffer to entire 50 uL; take an aliquot to run on a gel.
Digest (Lab 19)
Save remaining uncut PCR product.
DRAW the ligation and its products.
Lab 19: Cloning of lambda PCR product
Of course, blue/white screening allows discrimination of empty vector
PCR products are probably NOT fully digested
· As PCR progresses, the concentrations of reagents (esp. dNTPs) changes dramatically
· As dNTPs become “limiting”, Taq error rate increases
Also, fewer dNTPs increases the free [Mg] which also affects Taq
The undigested PCR products probably have mutations in the restriction recognition sequences, making them “undigestible”
· Consider consequences of this for cloning work: fragments lacking sticky ends will not be cloned; but other sequence errors are likely in final product
· See Internet activity on Pfu DNA polymerase (high fidelity)
Remind students to be very explicit in their lab notebooks about the cloning strategy:
What fragment was amplified by PCR;
Where the Bam & Hind sites are, and the size of the resulting fragment to be cloned;
What the vector looks like, both Bam & Hind in MCS
Sticky end ligation, single orientation, antibiotic selection and blue/white screening
Lecture notes: Alu PCR (Lab 22)
An example of a highly polymorphic locus in humans:
Within the tissue plasminogen activator (TPA) gene, an intron may or may not have an insertion of an Alu element
See picture p. 345 in text: Primers flank this Alu insertion site; if Alu element is present, will amplify a 400 bp fragment; if Alu is absent, 100 bp product. REMEMBER: You may be homozygous or heterozygous for this insertion.
Small fragments: we will use 2% agarose.
PCR can work with very small amount of starting material. Rinsing mouth to get a few cheek cells is adequate for this analysis.