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Cloning and gene expression in Pseudomonas denitrificans
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28 Aug 2010 - 19:10
Posts I've Made
In Topic: Ligation with small insert and large vector
Yesterday, 07:09 PM
Yes right. The competant cell efficiency is very important. And one more thing is the host organism.
In Topic: Ligation with small insert and large vector
14 May 2013 - 08:26 PM
http://www.insilico..../Lig_Input.html
In this ligation calculator you can try 1:5 or 1:10 ratio of Vector:Insert. The ratio of vector and insert depends on the size.
In this ligation calculator you can try 1:5 or 1:10 ratio of Vector:Insert. The ratio of vector and insert depends on the size.
In Topic: contamination possibility
13 May 2013 - 06:34 PM
Of course, there is possibility of contamination. Open -20 for long time causes the formation of ice on tubes but we cannot avoid that. If we need to search for something specific we need to keep open -20 sometimes. You can avoid by keep the tube on ice and wipe it before open.
In Topic: Sequencing problem and restricition enzyme digestion
07 May 2013 - 10:23 PM
Enzyme Cat# Temp Supplied NEBuffer Supplements % Activity in NEBuffer BSA SAM 1 2 3 4 BamHI* R0136 37°C NEBuffer 3 Yes No 75 100 100 100 EcoRI* R0101 37°C NEBuffer EcoRI No No 100 100 100 100
Double Digest Recommendation(s) for BamHI + EcoRI:
* BamHI has a High Fidelity version (R3136)
EcoRI has a High Fidelity version (R3101)
High Fidelity (HF) Restriction Enzymes have been engineered for reduced star activity
and have 100% activity in buffer 4 which may simplify your double digest.
Double Digest Recommendation(s) for BamHI + EcoRI:
- Digest inNEBuffer EcoRI + BSA at 37°C.
Note: BamHI may exhibit star activity in this buffer.
* BamHI has a High Fidelity version (R3136)
EcoRI has a High Fidelity version (R3101)
High Fidelity (HF) Restriction Enzymes have been engineered for reduced star activity
and have 100% activity in buffer 4 which may simplify your double digest.
In Topic: Sequencing problem and restricition enzyme digestion
07 May 2013 - 10:22 PM
Hi,
I think after sequencing your insert you can figure out. If the restriction site bases are wrong you may not get your target gene after digestion. The enzyme added to the reaction may cut the enzyme present in the pGEMT vector.
I think after sequencing your insert you can figure out. If the restriction site bases are wrong you may not get your target gene after digestion. The enzyme added to the reaction may cut the enzyme present in the pGEMT vector.
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