Paul.xu at 13:21, 7 August 2012

2012-08-07T13:21:32Z

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== LR Reaction to Transfer Genes to Expression Vector-- for 1 reaction ==

1. Measure the DNA concentration of both constructs (Donor and Destination) 

 

2. Calculate the volume in ul needed of ENTR plasmid (25 ng) ul needed = 25 ng needed / (concentration in ng/ul) 

 

3. Calculate the volume in ul needed of DEST plasmid (25 ng) ul needed = 25 ng needed / (concentration in ng/ul) 

Very Important: never use more than 25 ng Dest vector 

4. Prepare in an autoclaved tube 

*ENTR-vector (25 ng)  0.5 ul 
*DEST-vector (25 ng)  0.5 ul

 

5. Remove the 5 x LR-Clonase II Enzyme Mix from -20°C or  -80°C This is expensive stuff don't leave it to rot in the ice-bucket!

 6. Add '''0.25 ul''' of the 5 x LR-Clonase Enzyme II Mix to the Gateway reaction and mix well

 7. Store the enzyme mix and buffer '''immediately''' at -80°C !!! 

 

8. Incubate at '''room temperature''' for '''1 to  5 hours or O/N'''. 

 

9. Add 0.25 ul of Proteinase K solution and incubate for 10 min at 37°C

 10. Transform bacteria with entire reaction:

Mammalian expression vectors such as Adenovirus, Lentivirus and AAV vectors: STBL 3

Bacterial Expression Vectors: STBL3, Top10 or DH5alpha

 

11. Plate bacteria with proper antibiotic (Amp) selection. 12. Digestion, PCR or sequencing to verify clones

 

== LR Reaction to Transfer Genes to Expression Vector-- for 8 reactions ==

1. Measure the DNA concentration of both constructs (Donor and Destination) 

2.  Take out  5 x LR-Clonase II Enzyme Mix from  -80°C,  put it in dry ice. 

Prepare in an autoclaved tube 

*DEST-vector (200 ng):  4 ul 
*5 x LR-Clonase II Enzyme Mix: 2 ul

mix well by pipetting up and down for a few times

Note: LR Clonase II is very expensive stuff,  don't leave it to rot in the ice-bucket! Put it back to -80 °C immediately. 

3. Aliquot 0.7 ul to 8 tubes. 

4. Add 0.5 ul DONOR or ENTR vector with gene of interest.

 

5. Incubate at '''room temperature''' for '''5 hours or O/N'''.

6. Add 0.25 ul of Proteinase K solution and incubate for 10 min at 37°C

7. Transform bacteria with entire reaction:

Adenovirus, Lentivirus and AAV vectors: STBL 3

Bacterial Expression Vectors: Top10 or DH5alpha 

8. Plate bacteria with proper antibiotic (Amp) selection. 9. Digestion or sequencing to verify 

Do not use the following protocol so far.

== Invitrogen Protocol ==

1. Add the following components to a 1.5 ml tube at room temperature and mix: 

*Entry clone (50-150 ng) 1-7 µl
*Destination vector (150 ng/µl) 1 µl
*TE buffer, pH 8.0 to 8 µl

2. Thaw on ice the LR Clonase ™ II enzyme mix for about 2 minutes. Vortex the LR Clonase ™ II enzyme mix briefly twice (2 seconds each time). 3. To each sample, add 2 µl of LR Clonase ™II enzyme mix to the reaction and mix well by vortexing briefly twice. Microcentrifuge briefly. 4. Return LR Clonase ™ II enzyme mix to -20°C or -80°C storage. 5. Incubate reactions at 25°C for 1 hour. 6. Add 1 µl of the Proteinase K solution to each sample to terminate the reaction. Vortex briefly. Incubate samples at 37°C for 10 minutes 

== BP Reaction ==

Creating a Gateway entry clone from an attB-flanked PCR product is an easy 1 hour reaction. See below for an overview of the set-up. For more detailed information, refer to the manual.

1.Add the following components to a 1.5 ml tube at room temperature and mix: attB-PCR product (=10 ng/µl; final amount ~15-150 ng) 1-7 µl Donor vector (150 ng/µl) 1 µl TE buffer, pH 8.0 to 8 µl 2.Thaw on ice the BP Clonaseâ¢ II enzyme mix for about 2 minutes. Vortex the BP Clonaseâ¢ II enzyme mix briefly twice (2 seconds each time). 3.To each sample (Step 1, above), add 2 µl of BP Clonaseâ¢ II enzyme mix to the reaction and mix well by vortexing briefly twice. Microcentrifuge briefly. 4.Return BP Clonaseâ¢ II enzyme mix to -20°C or -80°C storage. 5.Incubate reactions at 25°C for 1 hour. 6.Add 1 µl of the Proteinase K solution to each sample to terminate the reaction. Vortex briefly. Incubate samples at 37°C for 10 minutes.

 

== Invitrogen BP Reaction ==

Creating a Gateway® entry clone from an attB-flanked PCR product is an easy 1 hour reaction. 

1. Add the following components to a 1.5 ml tube at room temperature and mix: 

*attB-PCR product (=10 ng/µl; final amount ~15-150 ng) 1-7 µl
*Donor vector (150 ng/µl) 1 µl
*TE buffer, pH 8.0 to 8 µl

2. Thaw on ice the BP Clonase™ II enzyme mix for about 2 minutes. Vortex the BP Clonase™ II enzyme mix briefly twice (2 seconds each time). 3. To each sample, add 2 µl of BP Clonase™ II enzyme mix to the reaction and mix well by vortexing briefly twice. Microcentrifuge briefly. 4. Return BP Clonase™ II enzyme mix to -20°C or -80°C storage. 5. Incubate reactions at 25°C for 1 hour. 6. Add 1 µl of the Proteinase K solution to each sample to terminate the reaction. Vortex briefly. Incubate samples at 37°C for 10 minutes. 

 '''Transformation'''

1.Transform 1 µl of each LR reaction into 50 µl of One Shot ® OmniMAX 2 T1 Phage-Resistant Cells (Catalog no. C8540-03). Incubate on ice for 30 minutes. Heat-shock cells by incubating at 42°C for 30 seconds. Add 250 µl of S.O.C. Medium and incubate at 37°C for 1 hour with shaking. Plate 20 µl and 100 µl of each transformation onto selective plates. Note: Any competent cells with a transformation efficiency of >1.0 x 108 transformants/µg may be used. 2.Transform 1 µl of pUC19 DNA (10 ng/ml) into 50 µl of One Shot ® OmniMAX 2 T1 Phage-Resistant Cells as described above. Plate 20 µl and 100 µl on LB plates containing 100 µg/ml kanamycin, or the appropriate selection marker for your donor vector.

'''''Expected Results''''' An efficient BP recombination reaction will produce >1500 colonies if the entire BP reaction is transformed and plated.

 

Gateway BP-Reaction II

*Design PCR-Primers with attB1.1 and attB2.1 sites
*Gel-purify you PCR product
*Make sure you have a PCR product with attB1.1 and attB2.1 and one DONR clone
*Measure the DNA concentration of both constructs
*Calculate the amount in ng needed of PCR product (25 fmol each): ng needed = (length of the PCR product in bp) x 0.0165
*Calculate the volume in µl needed of DONRTM plasmid (75 ng): µl needed = 75 ng needed / (concentration in ng/µl)
*Prepare in a new eppi the Gateway® reaction:  

{| cellpadding="2" border="0" style="width: 358px; height: 73px;"
|-
| style="width: 220px;" |
PCR-product

| style="width: 80px;" |
( ? ng)

|-
| style="width: 220px;" |
pDONRTM-vector

| style="width: 80px;" |
(75 ng)

|-
| style="width: 220px;" |
add water to a total volume of 2µl

| style="width: 80px;" |
 

|}

*Remove the 5 x BP-Clonase II enzyme mix from -20°C
*Pipett 0.5 µl of this BP-Clonase II enzyme mix to the Gateway® reaction
*Store the enzyme mix and buffer immediately at -20°C
*Incubate at room temperature over night
*Add 0.5 µl of Proteinase K solution and incubate for 10 min at 37°C (IMPORTANT)
*Transform DH5α bacteria

 

 

'''Materials needed''' 

BP-Clonase  II enzyme mix (# 11789-020) by Invitrogen 

'''Long Protocol:'''

'''1. Design PCR-Primers with attB1.1 and attB2.1 sites'''

Check out the protocol on primer design. In short - you should take care of some things. The Gateway® clones have a reading frame which should be kept. Design primers that the PCR product starts with a ATG and ends with a STOP-codon or the last aminoacid (if you want to make a fusion protein). <a href="http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi">Primer3plus</a> is a powerful tool helping you to pick primers with the right annealing temperature which should be 60°C. Try to avoid self similarity and other things as usual, but because you are very limited in the position of the primers (its start and stop), I only care about annealing temperature and give it a try. Then just add to the primer which binds the start codon the attB1.1-sequence at his 5' End . To the primer which binds the stop codon or the last aminoacid add the attB2.1-sequence at his 5' End . The open reading frame is indicated and you should change the last two NN to code for an aminoacid of your choice. Good luck for the PCR! Because of the long 5' overhang and the restrictions on picking the primers, getting the PCR to work can be tricky.

 Improved and more efficient att sites used to recombine into pDONR 221:

attB1.1 GGG-GCA-ACT-TTg-tac-aaa-aaa-gtt-gNN

attB2.1 GG-GGC-AAC-TTT-GTA-CAA-Caa-agt-tgN

 The original att sites used to recombine into pDONR 221:

attB1 GGGG-ACA-AGT-TTg-tac-aaa-aaa-gca-ggc-tNN

attB2 GGG-GAC-CAC-TTT-GTA-CAA-Gaa-agc-tgg-gtN

 The att sites used to recombine into pDONR P4-P1R:

attB4 GGGG-ACA-ACT-TTg-tat-aga-aaa-gtt-gNN

attB1 GGG-GAC-TGC-TTT-TTT-GTA-Caa-act-tgN

 The att sites used to recombine into pDONR P2R-P3:

attB2 GGGG-ACA-GCT-TTc-ttg-tac-aaa-gtg-gNN

attB3 GGG-GAC-AAC-TTT-GTA-TAA-Taa-agt-tgN

'''2. Gel-purify PCR product'''

Purification of the PCR-product is needed to get rid of smaller side-products, which remove primer-dimers which can result in false positive colonies. Remember that you want to clone DNA, so the cutting should be made on the weakest UV-light available and as fast as possible. And of course you NEVER make a picture of the gel before. Use the kid for gel-purification available in your lab.

'''3. Make sure the PCR product has attB1.1 and attB2.1 and one DONR vector'''

You need a PCR product with the attB1.1 and attB2.2 and the DONR vector MUST have attP1 and attP2 sites, or it will not work.

 The amount of plasmids is not soo important as in a multiple Gateway® reaction, because it is more efficient. If you want to optimize you can calculate equimolar amounts of both plasmids as described in the How to measure DNA. Here we use double the amount of DEST-vector, because most of the ones we use are round and about double the size of the ENTR clones.

'''4. Measure the DNA concentration of both constructs'''

The amount of ENTR is not so important as in a multiple Gateway® reaction, because it is more efficient. If you want to optimize you can calculate equimolar amounts of both plasmids as described in the multiple Gateway® protocol. Here we use double the amount of DEST-vector, because most of the ones we use are round and about double the size of the ENTR clones.

'''5. Calculate the amount in ng needed of PCR product (25 fmol each):'''

ng needed = (length of the PCR product in bp) x 0.0165

'''6. Calculate the volume in µl needed of DONR plasmid (75 ng):'''

µl needed = 75 ng needed / (concentration in ng/µl)

 The DONR-vector should be tested for low background colonies (due to a

mutated ccdB-gene) when transferred in DH5alpha-bacteria.

'''7. Prepare in a new eppi the Gateway® reaction:'''

{| cellpadding="2" border="0" style="text-align: left; width: 361px; height: 86px;"
|-
| style="width: 220px;" |
PCR-product

| style="width: 80px;" |
( ? ng)

|-
| style="width: 220px;" |
pDONRTM-vector

| style="width: 80px;" |
(75 ng)

|-
| style="width: 220px;" |
add water to a total volume of 2 µl

| style="width: 80px;" |
 

|}

'''8. Remove the 5 x BP-Clonase II enzyme mix from -20°C'''

It is most efficiently mixed by pipetting up and down, do not vortex.

'''9. Pipett 0.5 µl of this BP-Clonase II enzyme mix to the Gateway® reaction'''

This is expensive stuff don’t leave it to rot in the ice-bucket!

'''10. Store the enzyme mix and buffer immediately at -20°C !!!'''

The enzymes looses 50% activity after 15 freeze-thaw cycles. The advantage of BP-Clonase II is that it can be stored at -20 °C because it contains already the buffer.

This is expensive stuff don’t leave it to rot in the ice-bucket!

'''11. Incubate at room temperature over night'''

Incubation overnight will enhance the reaction ca. 5-10 fold. This

is especially important for PCR products over 5.000 bp.

'''12. Add 0.5 µl of ProteinaseK solution and incubate for 10 min at 37°C (IMPORTANT)'''

This step will enhance the reaction ca. 100 fold!!!!. This is different to the LR-reactions which are only enhanced 2 fold by adding the proteinase K.

'''13. Transform DH5α bacteria'''

Transform competent cells with 2.5 µl.

'''14. Plate bacteria with proper antibiotic selection'''

The resulting ENTR-vectors are kanamycin resistant.

Known Issues:

*The reaction is very efficient. You can obtain about 200 colonies of which about 95 % are correct.

*BP reactions work better with linear templates like PCR-products. If you want to use plasmids, linearize them first with a suitable restriction enzyme.

*The obtained plasmids are big. To check for correct clones digestwith Sty I and in parallel with EcoRI and Hind III. Compare the pattern of bands with the predicted band size to find the correct clones.

 

LR Reaction

Transferring your gene from a Gateway® entry clone to destination vector is an easy 1 hour reaction. See below for an overview of the set-up. For more detailed information, refer to the manual.

1.Add the following components to a 1.5 ml tube at room temperature and mix: Entry clone (50-150 ng) 1-7 µl ,Destination vector (150 ng/µl) 1 µl ,TE buffer pH 8.0 to 8 µl

 2.Thaw on ice the LR Clonase  II enzyme mix for about 2 minutes. Vortex the LR Clonase â¢ II enzyme mix briefly twice (2 seconds each time).

 3.To each sample (Step 1, above), add 0.5 µl of LR Clonase â¢II enzyme mix to the reaction and mix well by vortexing briefly twice. Microcentrifuge briefly.

 4.Return LR Clonase  II enzyme mix to -20°C or -80°C storage.

 5.Incubate reactions at 25°C for 1 hour.

 6.Add 1 µl of the Proteinase K solution to each sample to terminate the reaction. Vortex briefly. Incubate samples at 37°C for 10 minutes.

'''Transformation''' 

Follow the protocol as indicated for the BP reaction, except use the appropriate selection marker for the LB plates suited to your destination vector (typically 100 µg/ml ampicillin). '''Expected Results''' An efficient LR recombination reaction will produce >5000 colonies if the entire LR reaction is transformed and plated.

One Tube Format 

If you want to transfer your attB-flanked PCR product directly into an expression clone, you can easily combine the BP and LR reactions using the following protocol. This will potentially eliminate the transformation and DNA isolation of the Gateway® entry clone.

 

1.In a 1.5 ml microcentrifuge tube, prepare the following 15 µl BP reaction: attB DNA (50-100 ng) 1.0-5.0 µl attP DNA (pDONRâ¢ vector, 150 ng/µl) 1.3 µl BP Clonase II enzyme mix 3.0 µl TE Buffer, pH 8.0 add to a final volume of 15 µl

 2.Mix well by vortexing briefly and incubate at 25°C for 4 hours. Note: Depending on your needs, the length of the recombination reaction can be extended up to 20 hours. An overnight incubation typically yields 5 times more colonies than a 1 hour incubation. Longer incubation times are recommended for large plasmids (=10 kb) and PCR products (=5 kb).

 3.Remove 5 µl of the reaction to a separate tube and use this aliquot to assess the efficiency of the BP reaction (see below).

 4.To the remaining 10 µl reaction, add: Destination vector (150 ng/µl) 2.0 µl LR Clonase II enzyme mix 3.0 µl Final volume 15 µl 5.Mix well by vortexing briefly and incubate at 25°C for 2 hours. Note: Depending on your needs, the length of the recombination reaction can be extended up to 18 hours. 6.Add 2 µl of proteinase K solution. Incubate at 37°C for 10 minutes. 7.Transform 50 µl of the appropriate competent E. coli with 1 µl of the reaction. 8.Plate on LB plates containing the appropriate antibiotic to select for expression clones.

Assessing the Efficiency of the BP Reaction

1.To the 5µl aliquot obtained from One-Tube Protocol, Step 3, above, add 0.5 µl of proteinase K solution. Incubate at 37°C for 10 minutes. 2.Transform 50 µl of the appropriate competent E. coli with 1 µl of the reaction. Plate on LB plates containing the appropriate antibiotic to select for entry clones.

Gateway® Vector Conversion

Converting your favorite set of cloning vectors to Gateway® Technology is a fairly straightforward protocol, and will ultimately allow you to streamline your cloning and expression process. To convert your cloning vector to a Gateway® destination vector, you will:

1.Choose the appropriate reading frame cassette to use depending on your needs. 2.Linearize the vector you wish to convert with a restriction enzyme of choice. If you use a restriction enzyme that generates an overhang, you will need to blunt the ends. 3.Remove the 5' phosphates from the vector using calf intestinal alkaline phosphatase. 4.Ligate the reading frame cassette into your vector using T4 DNA ligase. 5.Transform the ligation reaction into One Shot® ccdB Survival Competent E. coli and select for transformants. 6.Analyze transformants. 

[[Category:Protocols]]

Gateway-Cloning - Revision history

Paul.xu at 13:21, 7 August 2012