Agroinfiltration methods (FWD)

1, Use the stab culture supplied as a starting material and inoculate 5 mI overnight culture
(28'C for agro):
-5 ml L-broth
-5 µI tetracycline (5 µl/ml final) (Depend on the constructs)
-5 µI kanamycin (50 µl/ml final)  (Depend on the constructs)

2, Use all the 1 ml of the overnight culture to inoculate 50 ml L-broth and grow overnight:
-50 ml L-broth
-50 µI tetracycline (5 µglml final)
-50 µI kanamycin (50 µglml final)
-500µI MES (10 mM final) [optional]
-I0 µI Acetosyringone (20 µM final)

3, Precipitate the bacteria and resuspend the pellet in solution containing:
-10 mM MgCI2
-10 mM MES
-100 µM Acetosyringone

4, Leave on the bench for 2 to 3 hours before agroinfiltration (or overnight).

5, Perform the infiltration with a 2ml syringe. Simply press the syringe (no needle) on the
underside of the leaf, and exert a counter-pressure with your finger on the other side.
Infiltrate the maximum number of leaves, avoid cotyledons.


From David Baulcombe's website

Small RNA Cloning Procedure (FWD)

Introduction

The small RNA cloning procedure is based on adapter ligation. The adapter oligonucleotides are used for priming reverse transcription and for defining the orientation and sequence of the cloned small RNAs. This protocol is isotope free, utilises unmodified small RNAs and is routinely used to characterise miRNAs and siRNAs from various plant tissues. The total nucleic acid (TNA) isolation is adapted from White and Kaper (1989). Our small RNA cloning protocol results from modifications of protocols originally published by the Tuschl, Bartel and Carrington groups (Elbashir et al., 2001; Pfeffer et al., 2003; Lau et al., 2001; Llave et al., 2002) and may be cited as Chappell et al., 2005.

Small RNA cloning protocol

The gel purified small RNAs are ligated directly to a non-phosphorylated 5’-adapter oligonucleotide using T4 RNA ligase. The ligation products are separated from the excess of 5’-adapter on a 15% denaturing polyacrylamide gel and are subsequently ligated to a 5’-phosphorylated 3’-adapter oligonucleotide with a blocked 3’-hydroxyl terminus. The final ligation products are separated from the excess of 3’-adapter and are subjected to reverse transcription and PCR amplification. The gel purified PCR products are digested with EcoRI and NcoI restriction enzymes and subsequently concatamerised using T4 DNA ligase. The concatamers are ligated into an EcoRI-NcoI digested cloning vector and then TOP10 cells are transformed with the recombinant plasmids. Individual colonies are screened for the size of concatamer inserts by PCR and selected PCR fragments are purified and submitted for sequencing. The small RNA sequences are extracted from the sequence manually or automatically using software tools (e.g., Staden Package or software developed in-house).

From :David Baulcombe's website

Perfect overlap PCR protocol from Nature

Perfect overlap PCR protocol from Nature


Nature Protocols 2, -924 - 932 (2007) 

Gene splicing and mutagenesis by PCR-driven overlap extension

Karin L Heckman¹ & Larry R Pease¹

ABSTRACT

Extension of overlapping gene segments by PCR is a simple, versatile technique for site-directed mutagenesis and gene splicing. Initial PCRs generate overlapping gene segments that are then used as template DNA for another PCR to create a full-length product. Internal primers generate overlapping, complementary 3′ ends on the intermediate segments and introduce nucleotide substitutions, insertions or deletions for site-directed mutagenesis, or for gene splicing, encode the nucleotides found at the junction of adjoining gene segments. Overlapping strands of these intermediate products hybridize at this 3′ region in a subsequent PCR and are extended to generate the full-length product amplified by flanking primers that can include restriction enzyme sites for inserting the product into an expression vector for cloning purposes. The highly efficient generation of mutant or chimeric genes by this method can easily be accomplished with standard laboratory reagents in approximately 1 week.

Fulltext 

http://www.nature.com/nprot/journal/v2/n4/full/nprot.2007.132.html

PCR Overlap Extension

This protocol from internet, it looks very good. here are some comments from me :)
4.2, I have used phusion, there is no problem for fragment under 3kb. I don't know why the author state this, I am not sure the reason. but one good point if you don't use phusion, is that you can do TA cloning after purification.

Overview

Create long DNA fragments from shorter ones. This method is also called "Splicing by Overlap Extension" or SOEing.

Procedure

1. Design Primers:
   1. These primers are like bridges between the two parts you want to assemble together.
   2. You will order two primers which are complements of one another.
   3. These primers will each have a 60°C Tm with one part and a 60°C Tm with the other part.
   4. The "end primers" will not have any complements and will likely only have restriction sites.
2. "Extension PCR" PCR amplify the necessary fragments separately
   1. Use a proofreading polymerase enzyme.
   2. Use an annealing temp of 60°C.
3. Clean up the product using a DNA column.
4. "Overlap PCR" Use cleaned up fragments as template in a PCR reaction:
   1. About 1/2 to 3/4 volume of the Overlap PCR reaction should be equimolar amounts of purified fragments.
   2. Do not use Phusion polymerase. Try Pfu Turbo.
   3. Do not add any primers; the templates will prime each-other.
   4. Run 15 PCR cycles without primers.
   5. Use an annealing temp of 60°C.
5. "Purification PCR" Add end primers to the Overlap PCR reaction:
   1. Continue cycling for another 15-20 rounds.
   2. Use an annealing temp of 72°C
6. Gel extract the correct size fragment.
7. Clone into the desired vector.
1. Digest
2. Ligate
3. Transform
4. Select
5. Sequence

From:    http://openwetware.org/wiki/PCR_Overlap_Extension