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PCR OF PHAGE FROM PSB STOCKS
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PCR protocol
20 ul PCR reactions (96 well PCR plates1)
The primary pick plate (contining 15ul phage/PSB) not the DMSO archive plate is used as DNA source.
All steps are carried out on ice.
PCR reaction mix per 20 ul reaction:
2.0 ul 10x Reaction Buffer (Mg free) 2
2.0 ul 2mM dNTP mix
1.2 ul 25mM MgCl2 3
0.4 ul primer (SKPL) 100ng/ml
0.4 ul primer (T7PL) 100ng/ul
2.0 ul phage/PSB
0.08 ml Taq (5000 units/ml)4
12 ul dH2O
_____
20.0 ul total vloume
Alternative primers that may be used to amplify inserts from Lambda Zap II and Lambda Uni Zap XR vectors
FORWARD PRIMERS (5')
M13R 5'AGCGGATAACAATTTCACACAGGA 3'
T3 5'ATTAACCCTCACTAAAG3'
SKPL 5' GGCCGCTCTAGAACTAGTGG 3'
REVERSE RIMERS (3')
M13L 5' CGCCAGGGTTTTCCCAGTCACGAC 3'
T7 5ACTATAGGGAGCTAAGCTTGG3'
T7PL 5' CTCACTATAGGGCGAATTGG 3'
SKPL and T7PL are our own primers which lie close to the EcoRI and XhoI restriction sites. These primers work well for amplifican of EcorI/Xho I inserts but T7PL cannot be used when amplifying EcoRI/XbaI inserts.
The positions of the primers are given relative to the sequence of the pBluescript Multiple Cloning Site below.
570 580 590 600 610 620
TAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAGCGCGCGTAAT
V G P N E W D R R Q L V V A L S R A Y Y
_________________________LAC Z ORF__________________________
____________________________MCS_____________________________
___M13 -20 PR____
___
________
_____M13 L PRIMER_20____
_________M13R BY M13L PRODUCT; 275 BASES IN WT__50_______
Xho_1
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630 640 650 660 670 680
ACGACTCACTATAGGGCGAATTGGGTACCGGGCCCCCCCTCGAGGTCGACGGTATCGATA
S E S Y P S N P V P G G R S T S P I S L
_________________________LAC Z ORF__________________________
____________________________MCS_____________________________
_____10_______
___T7 PLUS LONG __20
10________20__
_60________M13R BY M13L PRODUCT; 275 BASES IN WT__110_______
___XHO I / ECO R I_0__
_______
_______
Eco_R_1 Xba_I
| |
690 700| 710 720 730| 740
AGCTTGATATCGAATTCCTGCAGCCCGGGGGATCCACTAGTTCTAGAGCGGCCGCCACCG
S S I S N R C G P P D V L E L A A A V A
_________________________LAC Z ORF__________________________
____________________________MCS_____________________________
120________M13R BY M13L PRODUCT; 275 BASES IN WT__170_______
___XHO I / EC____
_10________20
_10________20________ECO-SAC FRAGMENT____50________60_______
________1_ECO-XBA FRAGMENT__30_______
___SK PRIMER_____
20_SK PLUS
LONG ____
750 760 770 780 790 800
CGGTGGAGCTCCAGCTTTTGTTCCCTTTAGTGAGGGTTAATTGCGCGCTTGGCGTAATCA
T S S W S K N G K T L T L Q A S P T I M
_________________________LAC Z ORF__________________________
____________________________MCS_____________________________
180________M13R BY M13L PRODUCT; 275 BASES IN WT__230_______
____T3 PRIMER______
___SAC PRIMER____
_70_ECO-SAC FRAGMENT_90__
__
810 820 830 840 850 860
TGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGA
T M
_______
____________________________MCS_____________________________
24_M13R BY M13L PRODUCT; 275 BASES____
___2_M13 R PRIMER_______
___10_________
870 880 890 900
GCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCT
_____________MCS_______________
1. Set PCR block5 onto preheat to 80°C
2. Defrost primary pick plate (containing remaining 15ml phage/PSB) not DMSO archive plate. Store on ice.
3. Prepare a master mix (MM) of reagents containing sufficient water, buffer, dNTP, MgCl, primers for the number of reactions. To PCR one 96 well PCR plate prepare a master mix for 110 reactions (to allow for pippetting errors).
In order mix:
220 ul 10x Reaction Buffer (Mg free)
220 ul 2mM dNTP mix
132 ul 25mM MgCl2
44 ul primer (SKPL) 100ng/ml
44 ul primer (T7PL) 100ng/ul
8.8 ul Taq (5000 units/ml)
Add Taq and mix reagents well.
5. Aliquot 18 ul PCR MM to each well of PCR plate (on ice).
6. Add 2 ul phage/PSB DNA from the primary pick plate to the MM in wells. Mix by gentle pipetting.
7. Cover reactions with drop of mineral oil
8. Seal plate.
9. Place PCR plate on prewarmed PCR block and begin cycle
PCR Cycle (program 15 on our touchdown)
1 cycle 94°C 15 sec 55°C 20 sec 72°C 3 min
35 cycles 72°C 10 min
1 cycle 4°C hold
Run 3 ul of each PCR reaction run out on 1.6% agarose gel to check inserts.
Our gel electorphoresis equipment 6 is compatible with the use of a multichannel pippete. Samples can be loaded on the gel directly from a microtitre plate and 96 samples can be run on a single gel.
You should expect to see only one PCR product per clone but a range of different sized PCR products between clones. Some clones may have been cross contaminated and will produce more than one PCR product these should not be selected for sequencing. In addition, some non recominant phage may have been picked and PCRed. The size of your non recombinant phage (wild type) PCR product will depend on the primers you used in your PCR. All clones containing cDNA inserts will have a PCR product larger than the wild type.
From your PCR products reject:
Clones with more than one PCR product.
Clones that have failed to amplify or amplified poorly to produce only faint PCR products.
Clones with small cDNA inserts <150bp.
The PCR products selected for sequencing should be regridded to a fresh microtitre plate for cleaning and sequencing.
Footnotes
1. Hybaid Omniplates. (cat order no HB-TR3-MT)
2. 10 reaction Buffer (part M190A)
3. 25 mM MgCl 2 supplied with Taq (part A351A)
4. Promega Taq DNA polymerase 5,000units/ml (Cat no. M1661)
5. Hybaid Touchdown machine (hot lid)
6. Gel electophoresis equipment from Hybaid.